Parasitizing Behavior of Ixodes Uriae Ticks on Chilean Magellanic Penguin (Spheniscus Magellanicus) and Their Importance As Pathogen Vectors

Parasitizing behavior of Ixodes uriae ticks on Chilean Magellanic penguin (Spheniscus magellanicus) and their importance as pathogen vectors

Johan Stedt

2009: Bi9

Degree project work in Biology Level: D

University of Kalmar School of Pure and Applied Natural Sciences 2009

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Parasitizing behavior of Ixodes uriae ticks on Chilean Magellanic penguin (Spheniscus magellanicus) and their importance as pathogen vectors

Johan Stedt, Biology 240hp

Degree Project Work, Biology: 30 hp

Supervisor: Assistant Professor, Jonas Waldenström School of Pure and Applied Natural Sciences Kalmar University

Examiner: Assistant Professor, Lars Riemann School of Pure and Applied Natural Sciences Kalmar University

Abstract Ticks are vectors for a larger number of viruses and bacteria than all other arthropod taxa, including mosquitoes. In Europe is it foremost Borrelia spirochetes and the Flavivirus Tick-borne Encephalitis virus that cause disease in humans. In this study, the tick species Ixodes uriae has been studied. I. uriae have a circumpolar distribution in both hemisphere and can be found both in Arctic and Antarctica. I collected ticks from Magellanic penguins in south Chile and analyzed them to see if they carry Borrelia spirochetes or Flavivirus. Totally were 218 ticks collected from 165 controlled penguins. All ticks were collected from adult penguins and the parasitizing ticks were all found in the auditory meatus which is a new phenomena compared to earlier studies. Both Borrelia spirochetes and Flavivirus were found in the collected ticks using PCR techniques. This is an interesting result since not much research has been performed in this geographical area before. Until date there is only one species of Borrelia (Borrelia garinii) found in I. uriae on the southern hemisphere and new Flavivirus is regularly found around the world. Unfortunately we have not been able to determine species of the Borrelia spirochetes or Flavivirus so far but this work will be continued.

Sammanfattning Fästingar är de artropoder som är bärare av högst antal arter av virus och bakterier, även myggor inräknat. I Europa är det främst flaviviruset Tick-borne Encephalitis virus och borrelios orsakat av Borrelia spirocheter som orsakar sjukdomsfall. Sett globalt finns dock ett stort antal olika patogener som kan ge oss sjukdomar. I denna studie har fästingar av arten Ixodes uriae insamlats från pingviner på ön Isla Magdalena i södra Chile. I. uriae kallas populärt för ”havsfågelfästingen” eftersom den i stort sett uteslutande parasiterar på olika typer av havsfågelarter. Arten har en cirkumpolär utbredning på både norra och södra halvklotet och finns i både Arktis och Antarktis. Detta märkliga utbredningsområde beror troligen på havsfåglarnas transekvatoriella flyttningsvanor. Det är sedan tidigare studier känt att I. uriae kan vara bärare av Borrelia spirocheter samt ett flertal virus, bland annat flera genera av Arbovirus. I denna studie har vi undersökt om fästingarna på Isla Magdalena bär på Borrelia spirocheter och Flavivirus. Sammanlagt insamlades 220 fästingar med åldersfördelningen 14 larver, 188 nymfer och 16 adulta. Av de 220 insamlade fästingarna hittades 218 i örongången på pingvinerna. Detta är en ny företeelse i jämförelse med andra studier genomförda på I. uriae. Tidigare studier har funnit fästingar på havsfåglarnas mjukdelar samt huvud och nacke. Halva fästingarna analyserades genom att vävnad odlades i ett speciellt medium, BSK II, för att försöka få tillväxt av Borrelia spirocheter. Återstående halvan av fästingarna användes för att detektera både Borrelia spirocheter och Flavivirus genom PCR tekniker. För att genomföra detta utfördes först en extraktion av totalt RNA, vilket sedan kunde göras om till cDNA. Detta cDNA kunde därefter användas för att med hjälp av optimerade primers utföra Real-tids PCR och därmed detektera eventuella Borrelia spirocheter och Flavivirus. Totalt detekterades Borrelia spirocheter i två fästingar medan Flavivirus detekterades i 30 fästingar. Detta är mycket intressanta resultat eftersom endast få liknande studier har genomförts inom detta geografiska område. Genom att artbestämma de positiva Flavivirus och Borrelia stammar som vi detekterat skulle man sannolikt ha möjlighet att diskutera kring vilka spridningsvägar som finns av fästingburna infektioner mellan den studerade fästingpopulationen och andra öar. Dessvärre har vi hittills inte lyckats artbestämma de positiva Flavivirus eller Borrelia stammarna. Detta arbete kommer att genomföras inom en snar framtid.

Table of contents 1. Introduction 6 1.1. Ticks 6 1.1.1. Taxonomy 6 1.1.2. Ecology 6 1.1.3. Ixodes uriae 7 1.2. Seabirds as reservoirs of pathogens 8 1.3. Ticks as a vector 8 1.3.1. Ticks as a vector 8 1.3.2. Borrelia-spirochetes 9 1.3.3. Arbovirus 10 1.3.4. Flavivirus 10 1.3.5. Ixodes uriae as a vector 11 1.4. Hosts in this studie 11 1.5. Related study 12 2. Material and method 12 2.1. Sampling site 12 2.2. Sampling 13 2.3. Analysis 14 2.3.1. Cultivation 14 2.3.2. RNA extractions and PCR procedures 14 2.3.2.1. Bead-beating (TissueLyser) 14 2.3.2.2. RNA extraction 15 2.3.2.3. RT-PCR 15 2.3.2.4. Real-time PCR 15 2.4. Positive samples 17 2.5. Statistical analysis 17 3 Results 17 3.1. Distribution of ticks on the island 17 3.2. Parasitizing 18 3.3. Pathogens 18 3.3.1. Borrelia 18 3.3.2. Flavivirus 19 4. Discussion 20 4.1. Life stages 21 4.2. Parasitizing 21 4.3. Pathogens 21 4.3.1. Borrelia 21 4.3.2. Flavivirus 22 5. Acknowledgements 22 6. References 23

Question we would like to answer concerning I uriae on Isla Magdalena:

Which species of ticks are possible to find on Isla Magdalena?

In which habitats on the island is it possible to find ticks?

On which body parts do the ticks infest the penguins?

Is it possible to detect Borrelia spirochetes or Flavivirus in the ticks?

Can we find the same genospecies of Borrelia as on other Islands?

1. Introduction 1.1. Ticks 1.1.1. Taxonomy Ticks are arthropods which mean that they belong to invertebrates and have an exoskeleton and a segmented body (Sonenshine 1991). Taxonomically ticks belong to the class Arcaria and are comprised in the suborder Ixodida (James & Oliver 1989). The suborder Ixodida is separated into three families, Argasidae, Ixodidae and Nuttalliellidae. The family Ixodidae is also called “hard ticks” and is the biggest family consisting of approx. 690 species of which approx. 250 species belong to the genus Ixodes (Sonenshine 1991). Ticks from the genus Ixodes can parasitize on humans and spread pathogens. These are the ticks we usually refer to as ”ticks” in common talk. For example the most common tick species in Sweden Ixodes ricinus belongs to this genus. Ticks from the Ixodes-complex are the most geographically widespread genus of the Ixodidae -family and are possible to find in all continents including the Antarctic regions (James & Oliver 1989).

1.1.2. Ecology Hard ticks have three life stages: larvae, nymphs and adults. To go from one life stage to the next the ticks have to moult. To accomplish a moult, ticks have to get a blood meal from a host. A host for a hard tick can be several types of organisms such as birds, mammals or reptiles, depending on both host availability and tick species (Olsen 1995). Hard ticks are slow feeders which mean that their blood meals take several days to complete (usually between 3-8 days) (Sonenshine 1991). In the adult stage, only the females parasitize for a full blood meal, this to be able to produce eggs. Adult males also use blood to feed but take only small meals for their survival. When the adult female has completed her blood meal she has increased her weight 80-120 times and is then able to produce a large amount of eggs (500-5000) before she dies (James & Oliver 1989). The incubation time for the eggs and also the whole lifecycle for a tick vary depending on tick species but also by temperature, humidity and host availability (Arthur 1968; James & Oliver 1989).

Different tick species have evolved different strategies to accomplish their life cycles and thereby use different amount of time. Most hard tick species use separate hosts between every life stage but some species have evolved a one-host strategy which mean that they are able to use the same individual as host in all stages (James & Oliver 1989). By using this strategy the ticks are able to complete their lifecycle in a shorter time (Sonenshine 1991). Despite this most hard ticks are three-host species. Some species are specialized to use only one species but different individuals as hosts. For example the tick Ixodes lividus only parasitize on one bird-species, the Sand martin (Riparia riparia). Most hard ticks have a broader spectrum of hosts and use different host species in each stage. Often a smaller organism is used as host during the larvae and nymph stage and a larger host in adult stage. I. ricinus, the most common tick species in Sweden often uses a small animal like a rodent or passerine bird in the larvae and nymph stage and a larger mammal, for example a deer in the adult stage. The habitat requirements are very different between tick species, but some aspects are similar across species. During the non parasitic phase ticks need a habitat with both suitable temperature and humidity to survive. At the same time it is necessary that the habitat offers good opportunities to find new hosts. Often these non-parasitic phases take place in caves, under stones or in bird nests (James & Oliver 1989). This offers good protection for predators and also protects them from unsuitable weather conditions. In this study we have used the tick species Ixodes uriae which is one of the most climate tolerant tick species.

1.1.3. Ixodes uriae I. uriae also called the seabird tick is one of the geographically most widespread tick species in the world and also one of the most climate tolerant species and has a circumpolar distribution in both hemispheres (Chastel 1988; McCoy et al. 1999). I. uriae is the only tick species resident in Artica and Antarctica, which implies that it is adapted to life in both dry and cold climates (Benoit et al. 2006). The tick species parasitize almost exclusively on different kind of seabirds, all together more than 50 different seabird species have been found as hosts (McCoy et al. 1999). On the southern hemisphere penguins (family Spheniscdae) seem to be the dominating bird family used as hosts and in the northern hemisphere auks (family Alcidae) have the same position (Olsen et al. 2003; Benoit et al. 2006). In rare cases mammals and humans have been confirmed to be infested by I. uriae (Mehl and Traavik 1983). The life cycle has a broad variation in length depending on geographic location. This is probably affected by factors like photoperiod, temperature and moisture, but an important factor is also availability of suitable hosts (James & Oliver 1989). In many seabird colonies the ticks only have a possibility to infest their hosts during the short breeding season when the birds are offshore for a longer period (Frenot et al. 2001). This has led to the fact that different populations of I. uriae have to adapt to different lifecycles depending on their host species (Frenot et al. 2001). Usually a lifecycle is completed in 4-5 years but in more extreme conditions as Antarctica the lifecycle can take up to eight years (Eveleigh & Threllfall 1974). In some seabird colonies the concentration of ticks can be extremely high and there have been studies indicating that

the parazitation on single hosts can be so intense that juvenile birds risk dying in hyperinfestation (Chastel et al. 1987; Bergström et al. 1996). Also the dispersal of I. uriae seems to be highly dependent of its host species (McCoy et al. 1999). Some seabird species like albatrosses and shearwaters migrate long distances during non-breeding season while species as penguins are more stationary around their breeding grounds. The global distribution of I. uriae is probably a consequence of dispersal from these long migratory seabirds (McCoy et al. 1999). However, many seabirds spend all their non-breeding time offshore and return to the same site during breeding season which should counteract dispersal. This could make dispersal of some tick population very limited and therefore give rise to local adaptations. Recent studies also indicate that populations of I. uriae often are highly species-specific which also can cause local adaptations (McCoy et al. 2005). Furthermore, a genetic study by McCoy et al. (2005) suggests that populations of I. uriae from the northern and southern hemisphere have so high genetic differentiation and are so isolated from each other that they in the future could be considered as separate species.

1.2. Seabirds as reservoirs of pathogens Seabirds often breed in huge colonies which can include several thousands to millions birds. This creates close contacts between the birds offering good opportunities for exchange of pathogens (Clifford 1979). During non-breeding season, seabirds perform long migrations both within and between the hemispheres. Since seabirds can perform fast migrations they can also act in the spread of ectoparasites such as ticks. This is probably one of the main reasons why I. uriae is circumpolarly distributed (Olsen 1995). Seabirds can act as a reservoir different micro-organisms. Identical strains of B. garinii have been found in ticks from seabird colonies in both hemisphere and most likely migrating seabirds have been acting as reservoirs in these cases (Gylfe et al. 2000). For example seabirds are suitable reservoirs for Borrelia spirochetes since they have a lower body temperature around 36-38oC compared to terrestrial birds that have a body temperature around 40o C (Warham 1990). Around Isla Magdalena where this study was performed there was large numbers of Arctic terns. This bird species perform one of the most long distant migrations between breeding and wintering grounds. The Arctic terns breed in the Northern Hemisphere and spend the winter in the Southern Hemisphere. Other seabird species, like black browed albatrosses, are long distance migrants and could be involved in transmission of various tick borne and other pathogens.

1.3. Ticks as vector 1.3.1. Ticks as vector Ticks are vectors for a large number of viruses and bacteria and are vectors to more kinds of microorganisms than all other atrophod taxas, including mosquitoes (James & Oliver 1989). Several tick species also have the ability to transmit these pathogens to humans during parazitation (James & Oliver 1989). Almost all tick-borne diseases are zoonotic, meaning that they can maintain in natural cycles without involving humans (Mather & Ginsberg 1994).

In Europe there is especially TBEV (Tick-borne Encephalitis virus) and Lyme disease caused by Borrelia spirochetes that are pathogenic to humans. However, also numerous other viruses, pathogenic to humans have been isolated from ticks, foremost different Flaviviruses (Chastel 1988; Major et al. 2009). Ticks can be infected with virus or bacteria when they feed on blood from a viremic/bacteremic host but it is also possible for a tick to become infected when they co-feed close together on a non-viremic host (Gritsun et al. 2003). It is also known that some pathogens can be transferred transovarially from the female to her offspring (Olsen et al. 1995).

1.3.2. Borrelia-spirochetes Borrelia is a bacteria of the order Spirochaetales, producing motile, helical shaped spirochetes which are 20-30 nm long. Their growth optimum is at 34-37o C, which makes many mammal and bird species available as hosts (Barbour 1984). Penguins, which are the hosts used in this study, have a body temperature of 36-38 o C which makes them well suited for growth of Borrelia spirochetes (Warham 1990). Borrelia spirochetes have no free-living stage and are therefore closely associated with Ixodes-ticks as a vector to complete their lifecycle (Olsen 2007). Borrelia spirochetes are often divided into three groups, Lyme disease Borrelia, relapsing fever Borrelia and animal spirochetes agents (Olsen 2007). The Borrelia agents that cause Lyme disease are referred to the bacterial complex Borrelia burgdorferi sensu lato. This group includes thirteen different genospecies: B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. japonica, B. andersonii, B. tanukii, B. turdi, B. valaisiana, B. lusitaniae, B. bissettii, B. sinica, B. californiensis and B. spielmanii (Duneau et al. 2008). Of these thirteen genospecies four are proved to be able to cause Lyme disease in humans (B. burgdorferi sensu stricto, B. garinii, B. afzelii and B. spielmanii) (Duneau et al. 2008). In Western Europe I. ricinus is the main vector for Borrelia spirochetes while in North America Ixodes pacificus has the same role. Totally more than 25 Ixodes-species are confirmed as vectors to Borrelia spirochetes (Eisen and Lane 2002). These tick species includes I. uriae that is confirmed to be a vector for three genospecies of Borrelia (Olsen et al. 1993; Duneau et al. 2008). The enzootic cycle of Borrelia spirochetes in I. uriae probably includes I. ricinus in the northern hemisphere since there are islands where it has been concluded that I. uriae and I. ricinus coexist and use the same host species. In these places there could probably occur an exchange through host species (Olsen 1995). Birds and mammals play an important roll in maintaining the enzootic Borrelia cycle. It is known that birds can act in several ways to maintain the cycle. Most important is that some bird species can act as reservoirs for spirochetes and in this way spread the bacteria back to new ticks. At the same time birds are able to transport parasitizing ticks during there migration and in this way spread infected ticks to new areas (Olsen 2007). What is known so far, the spirochetes have no significance as disease agents in wild birds (Olsen 2007). It is also known that many species of rodents and insectivores are able to act as reservoirs (Tälleklint & Jaenson 1994).

The distribution of Lyme disease ticks seems to correlate with the distribution of Ixodes- ticks on the northern hemisphere, which includes US in the west to Japan and Korea in the east (Olsen 1995). From the southern hemisphere there is still no confirmed cases of Lyme disease but B. garinii infected ticks have been detected in I. uriae (Olsen 1995).

1.3.3. Arbovirus The name Arbovirus comes from arthropod-borne virus, meaning that they are transmitted by arthropods, including mosquitoes, ticks and flies (McLean & Ubico 2007). In spite of the name there are several Arboviruses that are not transmitted by Arthropods and also several with unknown vectors (McLean & Ubico 2007). Arboviruses have a global distribution and are separated in twelve different genera (Major et al 2009). Totally around 600 different Arbovirus are registered and around 70 of these have been isolated from birds (McLean & Ubico 2007). There are numerous Arbovirus that have been sequenced, but there are still only a few that have been studied extensively in the Antarctic regions and only a very few sequences are so far available from the Antarctic region (Major et al 2009). In this study we have focused on Flavivirus.

1.3.4. Flavivirus Flavivirus are single-stranded RNA-virus with a genome of about 11,000 base pairs (McLean & Ubico 2007). The virus is composed of three structural proteins and seven non-structural proteins (McLean & Ubico 2007). The Flavivirus genera include around 70 different virus species and about half of them are associated with human disease (Ludwig & Iacono-Connar 1992). Flavivirus exist in all continents but each virus has a distinct geographic distribution, influenced by factors such as ecological habitats and distribution of both invertebrate and vertebrate hosts (Gould et al. 2001). Flavivirus can be both arthropod-vectored viruses and non-vectored viruses and are often separated into three classes, mosquito borne, tick-borne and where vector is unknown (Gould et al. 2001). So far, there are 14 flaviviruses that are known to be tick-borne (Table 1). The most common and also the most pathogenic tick-borne flavivirus is TBEV that can cause neuroinfections in humans. In Europe and Asia TBEV cause approximately 11 000 human cases per year (Gritsun et al. 2003). All tick-borne Flavivirus have similar replication strategies and are also biochemical similar even if they have a wide variation in virulence and ecology (Ludwig & Iacono-Connar 1992). Tick-borne Flavivirus is further separated in two distinct groups, mammalian virus and seabird virus (Gritsun et al. 2003). In the seabird group which is the most interesting group in this study there are three, possibly four known Flavivirus: Quleniy virus, Tyuleniy virus, Saumarez Reef virus, and Meaban virus (Gritsun et al, 2003). Seabird Flavivirus have been isolated from both Ixodes-ticks and Ornithodorus-ticks and are highly associated with seabirds (Ludwig & Iacono-Connar 1992). The virus in this group are widespread and isolation have been done from both the northern hemisphere (Tyuleniy virus and Meaban virus) and Australia in south hemisphere (Saumarez Reef virus) (Ludwig

& Iacono-Connar 1992).

Table 1. Tick-borne Flavivirus separated in Mammalian tick-borne virus group and Seabird tick-borne virus group. Virus species are written in normal style and subtypes in italic style (Gould et al. 2001).

Tick-borne flaviviruses Mammalian tick-borne virus group Louping ill virus Irish subtype British subtype Spanish subtype Turkish subtype Tick-borne encephalitis virus European subtype Siberian subtype Far Eastern subtype Omsk hemorrhagic fever Langat virus Kyasanur Forest disease virus Karshi virus Royal Farm virus Powassan virus Gadgets Gully virus Seabird tick-borne virus group Quleniy virus Meaban virus Saumaraez Reef virus Kadam virus

1.3.5. Ixodes uriae as a vector The tick species I. uriae is known to be a vector to several viruses (Chastel 1988; Major et al. 2009). There is several different genera of Arbovirus that have been isolated from I. uriae including Flavivirus, Nairovirus, Phlebovirus and Orbivirus (Major et al. 2009). Also Borrelia spirochetes from three genospecies have been found in I. uriae (B. burgdorferi sensu stricto, B. garinii and B. lusitaniae) (Duneau et al. 2008). Borrelia spirochetes from the genospecies B. garinii where first isolated from I. uriae on the island Bonden in north-eastern part of Sweden.This is also the only Borrelia genospecies so far found on the southern hemisphere (Olsen et al, 1993 & Olsen et al, 1995). More recent studies have indicated that Borrelia spirochetes are more widespread in I. uriae than it was thought before (Duneau et al, 2008).

1.4. Hosts in this study Magellanic penguin (Spheniscus magellanicus) is a relatively small penguin about 70 cm tall (Fig 1.). Magellanic penguins are common on breeding sites in South America and the world population is estimated to be between 4 500 000 and 10 000 000 birds (Del Hoyo et al. 1992). The breeding area

includes both the Atlantic and Pacific side of South America from central Chile and central Argentina in north down to Cape Horn (Del Hoyo et al. 1992). The penguins breed in dense colonies using burrows that are often less than one meter from each other. During the non-breeding season, in the Austral autumn, the birds migrate northwards from their breeding grounds to the ocean outside Brazil where they are pelagic (Davis & Darby 1990; Del Hoyo et al. 1992). During this time of the year Magellanic penguins have been found far from their breeding grounds and there are records from both Australia and

New Zeeland (Del Hoyo et al. 1992). Figure 1. Magellanic penguin (Spheniscus magellanicus) The breeding season begins in October when two eggs are laid and in late January or February the chicks are fledged. At this time the adult birds starts to moult, which continues until March when the migration starts (Davis & Darby 1990). This means that they spend around half of the year on the ocean.

1.5. Related study A related study on pathogens from I. uriae has recently been done by Major et al. (2009) on Macquarie Island (54o30oS, 158o55 o E) located between Australia and Antarctica. In this study I. uriae ticks were collected from the ground close to one King penguin (Aptenodytes patagonicus) colony and one Rockhopper penguin (Eudyptes chrysocome) colony. Virus found in the ticks were isolated in cell cultures and then sequenced. The analysis resulted in virus belonging to four of the 12 Arbovirus genera (Flavivirus, Orbivirus, Phlebovirus, and Nairovirus). The Flavivirus found were sequenced using primers based on Saumarez reef NS5- gene and E protein (Major et al. 2009). The result from the DNA-sequence was nearly identical to the so called Gadgets Gully virus isolated on Macquarie Island in the 1970´s. This Flavivirus does not group with other seabird flavivirus which indicates that I. uriae can be a vector also to mammalian Flavivirus.

2. Material and method 2.1. Sampling site Ticks for this study were collected during a Chile expedition in January 2009. The sampling took place on Isla Magdalena (52o55oW 70o34oS) in the Magellan sound southeast of Punta Arenas (Fig. 2). The island which has an area of 97 hectare is dry and mostly covered with sand and in some parts short

grass vegetation. On the island there is one resident sentinel living and during the penguins breeding season tourists visit the island daily. On the Island there is a large colony of Magellanic penguins estimated to around 60 000 breeding pairs and with juvenile birds included the population is around 200 000 birds. The Magellanic penguins breed close together all over the island and on the Island there is also high numbers of breeding Kelp gulls (Larus dominicanus) and Chilean skuas (Stercoraris chilensis). Also long-distance migratory birds like Artic terns (Sterna paradisaea) and black-browed albatrosses (Diomedea melanophris ) are roosting in high numbers around the island. During our two days on the island we recorded around 20 bird species. No terrestrial mammals are resident on the island.

Figure 2. Map showing South America and the location of Isla Magdalena (Map from Googlemaps).

2.2. Sampling Parasitizing ticks were collected from Magellanic penguins caught in and around their nests. Ticks were only collected from adult penguins since we had no license to catch juveniles. All body and also soft parts of the penguins were examined carefully to find attached ticks. To have the possibility to compare different habitats birds were caught both in dry areas on the island and in more moist and grassy areas.

2.3. Analysis The collected ticks were transported to Sweden in Eppendorf-tubes when they were still alive. Almost all ticks were still alive after the arrival to Sweden but 20 individuals had died and where therefore excluded from further analyses. The first part of the work took place at Umeå University where determination of species and life stage for each tick was settled. To definitely know that our collected ticks were of the species I. uriae some ticks were sent to Uppsala University for identification. Some ticks were also photographed to confirm the species identity. The ticks were then prepared for detection of Borrelia spirochetes and a broad range of Flavivirus. To perform these analyses each tick was washed in 70 % ethanol to remove ectobacteria from their shield. Directly after, the tick was cleaned in PBS (Phosphate-buffered saline) and then dried on a paper tissue. The ticks were then placed on a sterile glass slide where they were divided into two halves with a sterile scalpel. To avoid contamination a new glass slide and scalpel were used for every tick. One half of the tick were then placed in a 1.2 ml Eppendorf-tube with BSK II medium (Barbour 1984) for cultivation of Borrelia spirochetes and the second part were placed in a 1.5 ml Eppendorf tube and frozen in –80o C for detection of both Borrelia spirochetes and Flavivirus using PCR methods. The PCR procedures were performed at Department of Clinical and Experimental Medicine and Division of Medical Microbiology at Linköpings University. Here there are protocols optimized for screening of both Borrelia spirochetes and a broad spectrum of Flavivirus. In Linköping University there was also a possibility to use robots to perform the analysis which meant that there was a minimal manual liquid handling with the samples. This minimizes the risk of random variation in the samples.

2.3.1. Cultivation To cultivate Borrelia spirochetes the tick tissue was placed in a BSK II medium including around 20 ingredients foremost rabbit serum but also the antibiotics sulfamethoxazol and phosphomycin to inhibit growth of other bacteria species (Barbour 1984). The cultures were then incubated in 35o C for 21 days. Both after 72 h and after 21 days each culture was carefully examined for Borrelia spirochetes using a binocular microscope. Totally were tissue from198 ticks cultivated.

2.3.2. RNA extractions and PCR procedures 2.3.2.1. Bead-beating (TissueLyser) To make it possible to extract RNA from the ticks they were first homogenized. This was done by placing each tick in a tube with a 5 mm steel bead and 450μl lysis buffer, including a RNA extraction kit. At the same time 48 tubes could then be bead-beated at 25 Hz for 2 minutes in a Tissue Lyser (QIAgen). The product was then centrifuged for 3 minutes at 20 000 g and 400μl of the supernatant which included the RNA were transferred to a new tube by manual pipetting.

2.3.2.2. RNA extraction To extract total RNA from the homogenized tick-tissue a robot (Biorobot M48 Workstation (Qiagen)) and an extraction kit, Magattract RNA Tissue Mini M48 kit was used. Here there was possible to process 48 samples at the same time. Therefore 46 samples with tick-tissue and one positive and one negative control was used in each extraction. The positive control included 5μl Encephur vaccine injection fluids, containing inactivated TBEV, and 5μl of a Borrelia burgdorferi sensu stricto B31 strain. In the negative control no template was added. This resulted in extracted RNA eluted in 50μl RNase- free water. During one extraction the operation was disrupted which resulted in that RNA was extracted from totally 187 ticks.

2.3.2.3. RT-PCR In next step the extracted RNA was reverse transcribed to cDNA (complementary DNA) using the kit “illustra Ready- To-Go RT-PCR Beads” (GE Healthcare). This kit contains primers and a bead. The beads contain Taq DNA polymerase, Tris-HCL, KCl, MgCl2, dNTPs, Moloney Murine Leukemia Virus (M-MuLV) Reverse Transcriptase (FPLCpure™), RNAguard™, Ribonuclease Inhibitor (porcine) and stabilizers. To each transcription 10μl of the extracted RNA from each sample was used and all pipetting steps were done with a CAS-1200 pipetting robot. First the primers was added to the RNA and the tubes were placed in a thermal cycler (2720 thermal cycler, Applied Biosystems, Carlsbad, CA) and heated to 97o C for 5 minutes to avoid secondary structures in the RNA and to eliminate RNAses. The beads that had been dissolved in RNA-free water were then added to each sample and after that the tubes where incubated in 42o C for 30 minutes for reverse transcription and then heated to 95o C for 5 minutes to denaturate the reverse transcriptase. The more stable cDNA could then be frozen in -80o C until the real-time PCR was performed.

2.3.2.4. Real-time PCR The cDNA was then used to detect Flavivirus and Borrelia spirochetes from the samples using Real- time PCR. For Flavivirus SYBR Green technique was used while a LUX real-time PCR assay was used for detection of Borrelia spirochetes.

Flavivirus For detection of Flavivirus a pair of primers (PF1S and PF2R-bis) designed by Moureau et al. (2007) was used (Table 2 & 3). This primer pair enables detection of 51 different flavivirus species and probably there is possible to detect even more and also new flavivirus (Moureau et al. 2007). The reason that these primers have such a broad spectrum is that the Ns5-gene has highly conserved regions in all tested Flavivirus. To perform the Real-time PCR for detection of Flavivirus a Rotor-Gene Real-Time Analysis Software 6.0 was used with SYBR Green as a fluorescent reporter. In the Rotor-Gene 72 samples can be analysed at the same time. Therefore 32 ticks were prepared at the time and two duplicates of each sample were done together with one negative and

three positive controls. As positive controls ticks spiked with inactivated Flavivirus from three species used, Dengue fever virus, West Nile virus and Yellow fever virus. Also the positive controls used already in the extraction were used to confirm that earlier steps had worked properly. To the heat cycle the protocol optimized by Moureau et al. 2007 was used. This have the cycle conditions 50o C for 50 min, 95o C for 15 min followed by 40 cycles consisting of 94o C for 15 s, 50o C for 30 s, and 72o C for 45 s. During slowly rising of temperature from 60o C to 95o C the analyses of the melting curves was performed. The detection limit when using this protocol have a broad range for different flavivirus ranging from 2 to 20 500 copies per sample (For list see Moureau et al. 2007). Since the broad spectrums primers used forms a high amount of primer dimers due to complementarities between them we had to use the melting curves to separate primer dimers from positive samples. This was possible since Flavivirus have a dissociation temperature at 78.9-84.1o C while the primer-dimers have a dissociation temperature on <74o C (Moureau et al, 2007). From the samples where suspected positive flavivirus were found a second Real-time PCR was done. Samples that where positive even this time where count as positive and send for sequencing.

Borrelia For detection of Borrelia spirochetes a LUX real-time PCR assay was performed in an ABI PRISM 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). The pair of primers used was designed in Linköping by Wilhelmsson et al. (2009) (Table 2 & 3). These primers pair target a 131-base pair long sequence specific for the genus Borrelia and are tested for detection of the following Borrelia species: B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. recurrentis, B. duttonii, B. turicatae, B. hermsii, B. japonica and, B. miyamotoi. In each reaction 46 tick-samples with duplicates and 1 positive control were analyzed using a 96-well reaction plate. Also the positive controls used already in the extraction were used here to confirm that all steps had worked properly. To prepare each samples 20 µl reaction mix were used including 10 µl Platinum® qPCR SuperMix UDG (Invitrogen, Carlsbad, CA), 0.04 µl Rox reference (Invitrogen), 0.4 µl LUXTM Bor16SFL primer (10µM), 0.4 µl unlabelled Bor16SR primer (10 µM) (Invitrogen Corporation, Paisley, GB), 4.16 µl RNAse free water and 5 µl cDNA from the samples. All pipeting steps were also here done in the CAS-1200 pipeting robot. After the pipeting steps the samples were centrifuged at 900 g for 5 min before the Real-time PCR assay. During the Real-time PCR reaction the protocol optimized by Wilhelmson et al, (2009) was used following the cycle conditions, 50 °C for 2 min, 95 °C for 2 min, and then 45 cycles of 95 °C for 15 s, 58 °C for 30 s and, 72 °C for 30 s.

Table 2. Primer pairs with target region and amplicon length. Amplicon Primer pair Use length (bp) Target region PF1S-PF2R-bis Amplification of Flavivirus 269-272 Ns5 gene Amplification of Borrelia Bor16SFL-Bor16SR spirochetes 81-130 16s rRNA-gene

Table 3. Primer sequences and amplication position. Bor16SFL primer is labeled with fluorophore 6-carboxyfluorescein Genome Primer name Sequence (5´ 3´) position

PF1S TGY RTB TA Y AA C ATG ATG GG 8869-8888

PF2R-bis GTG TCC CA I CCNGCN GTR TC 9121-9140

Bor16SFL GAC TCG TCA AGA CTG ACG CTG AGT C 465347-465366

Bor16SR GCA CAC TTA ACA CGT TAG CTT CGG TAC TAA C 465447-465477

2.4. Positive samples From samples regarded as positive Flavivirus in the PCR the two duplicates was pooled and then vacuum centrifuged for 4 h in 30o C to concentrate the DNA. The product was then dissolved with 25µl TE-buffer and sent to Macrogen Inc. (Seoul, Korea) for sequencing. For positive Borrelia samples primers have to be optimized before sequencing could be done. The primers used in earlier studies have been used for DNA and target an rRNA intergenic spacer and could therefore not be used for the extracted RNA. This work is in progress and could not be presented here.

2.5. Statistical analysis All Statistical calculations were performed in the software SPSS Version 14.0. To compare prevalence between life stages and the frequency of parasitizing ticks in different habitats Fisher’s exact test was used. All p-values < 0.05 were considered significant.

3. Results 3.1. Distribution of ticks on the Island Totally were 218 ticks collected from 51 penguins (Mean: 4.27, SD=5.3, range1-26)(Fig. 3) and 216 of these ticks were collected from grassy habitats. Of the collected ticks were 6.4 % (n: 14) larvae, 86 % (n: 188) were nymphs and 7.4 % (n: 16) were adults. There was a statistically significant difference in frequency of parasitized penguins when different habitats on the Island were compared (Fisher’s exact test p< 0.001). In grassy habitats 75 % (49 of 65) of the controlled penguins had ticks compared to 2 % (2 of 100) in more arid habitats. Since

the sampling took place during breeding season the birds were stationary around there nests except during their fishing to have food for the juveniles. Despite that high numbers of parasitizing ticks were collected from the penguins in the grassy habitats no ticks could be found on the ground or in the nests in either habitat.

10 Number ofpenguins Number 5

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Parasitizing ticks/bird

Figure 2. Frequency distribution graph showing number of parasitizing ticks per penguin.

3.2. Tick sampling Of the 218 collected ticks were 216 found in the auditory meatus on the penguins. The remaining two ticks were found from base of the bill and in the neck. Despite the high concentration to auditory meatus all soft parts and also the plumage was examined as carefully as possible on every bird.

3.3. Pathogens 3.3.1. Borrelia When the ticks were cultured in BSK II medium there was not possible to find any Borrelia spirochetes in the samples. From the second part of the ticks RNA extractions and PCR procedures was performed in a try to detect Borrelia spirochetes. Here two samples strongly indicated that the sample was positive during Real-time PCR. Two more samples have indications to be positive during the Real-time PCR but need to be confirmed by sequencing since the exponential growth exceeded the threshold during the last cycles.

3.3.2. Flavivirus During the Real-time PCR, 30 samples had melting curves with a marked dissociation top between 78.9-84.1o C and were thereby treated as positive Flavivirus even if the virus concentration seemed to be low (Fig. 3). Totally as much as 16 % (30/187) of the analysed ticks were treated as positive for Flavivirus. When separating positive ticks after life stage, prevalence for each stage was 8 % (1/12) for larvae, 14 % (23/160) for nymphs and 43 % (6/14) for adults. There where no statistical difference in prevalence between juveniles and nymphs (Fisher’s exact test, p=0.44). Between nymphs and adults there was a difference suggested but it was not statistically significant (p=0.077). There was not possible to find any connection between infected ticks and individual penguins. Even if one penguin had three ticks positive to Flavivirus all other positives were scattered between different penguins. The positive samples were send for sequencing but there was not possible to receive any sequences because of primer mismatching aspects. Therefore new cDNA have to be prepared in a try to sequence them.

Figure 3. Dissociation curves with melting temperature of amplicons. Positive control (solid line), Negative control (dashed line) and sample treated as positive (dotted line).

4. Discussion This studies primary aim was to analyse the ticks for Borrelia spirochetes and Flavivirus to investigate whether the ticks act as vectors. This gave interesting results since I could detect both Borrelia spirochetes and Flavivirus in the ticks. Further we could observe that ticks were not evenly distributed over the island, instead they were restricted to certain habitats. Another interesting insight was that ticks were only using auditory meatus for parasitizing, which is a new phenomenon.

4.1. Life stages A higher number of nymphs compared to adults were found parasitizing on the penguins. This was expected, since there is high mortality between the tick life stages, and in the adult stage only females parasitize. It was more surprising that so few larvae were found compared to nymphs. Perhaps the juvenile ticks were so small that they were easily missed during examination of the penguins. It is also possible that juvenile ticks use juvenile penguins as hosts more often, since earlier life stages more often chose a smaller host to parasitize from (Olsen 1995).. Unfortunately, we had no possibility to control this theory since we had no license to catch the juvenile penguins. It could also be, as suggested by Bergström et al. (1999), that the ticks of different life stages have separated activity peaks and the larvae therefore was in a non-parasitic phase during our visit. Earlier studies regarding I. uriae have found single seabirds infested by hundreds of ticks (Chastel et al. 1987; Bergström et al. 1996). In this study the highest number found on one bird was only 26 ticks. This could maybe be explained by the fact that the ticks were only using the auditory meatus as feeding site. On the bird infested with 26 ticks the auditory meatus was full of ticks and there was no space for more. Since I. uriae has adapted so they have their parasitizing peak during their host’s incubation time, we could have missed this peak and only seen a small fraction of the parasitizing ticks.

4.2. Parasitizing In moist, grassy habitats on the Island, ticks were collected from 75 % (49/65) of the examined penguins, while almost no ticks were found in arid habitats of the island. This indicates that it is only in the more moist habitats that the ticks are able to survive during their non-parasitic stages. The non- parasitic stage is critical since the ticks then spend long periods moulting and during this time they need both protection and suitable humidity (Benoit et al. 2007). All parasitizing ticks except two were found in the auditory meatus on the adult penguins. This is a new phenomenon compared to earlier studies of I. uriae parasitizing adult penguins and other adult seabirds. Earlier studies have found parasitizing ticks mainly on the birds’ feet, head and neck ( Gauthier-Clerc et al1998; Bergström et al. 1999 & Mangin et al. 2003). That I. uriae on Isla Magdalena only use the auditory meatus could maybe reflect a local adaptation for survival when the penguins are under water. Since the penguins probably close there auditory meatus when diving this could protect the ticks from drowning. It could also be a local adaption to the arid environment since other bare parts of the penguin like feet and beak are too exposed to the sun. I. uriae populations are often strongly associated with one host species and adapt to that even when several seabird species are breeding on the same Island (McCoy et al. 2001). This offers good opportunities to adapt behaviourally after that host species. McCoy et al. (2001) showed that genetic differentiation between tick populations of sympatric black-legged kittiwakes (Rissa tridactyla) and Atlantic puffins (Fratercula arctica) was much greater than that between allopatric populations of either host. Also, I. uriae parasitizing on King penguins and Macaroni penguin (Eudyptes chrysolophus chrysolophus) breeding on the same island, Possession Island (Crozet Archipelago) in the Southern ocean, have adapted so that the two populations parasitize in different cycles depending on which host they use (Frenot et al. 2001). In the King

penguin colony the ticks completed their life cycle in three years while it was completed in two years in the Macaroni penguin colony (Frenot et al. 2001). By support from these studies it might be possible that the behaviour by only using the auditory meatus for parasitizing is a new and local phenomenon that this tick population have adapted. If the ticks are protected from drowning by using the auditory meatus this could also have the advantage that they are not so dependent to infest the penguins during their incubation time and therefore could perform their lifecycle in shorter time.

4.3. Pathogens 4.3.1. Borrelia Recent studies have indicated that Borrelia spirochetes probably have higher prevalence and diversity in I. uriae than previously assumed (Duneau et al. 2008). Most studies have been performed in the Northern hemisphere, where three genospecies from the complex B. burgdorferi sensu late have been isolated. The marine Borrelia cycle is therefore highly interesting and needs more attention not least in the Southern hemisphere. Studies have so far detected Borrelia spirochetes from only some locations on the southern hemisphere. The large geographic area of distribution is most likely caused by seabirds’ long migratory routes. Most seabirds migrate within one hemisphere, which makes transhemispheric spread more unusual. That transhemispheric migration is uncommon could be a reason that only B. garinii so far has been found from I. uriae on the Southern hemisphere. However, it is not impossible that transhemispheric exchange of Borrelia spirochetes occur, which is suggested from earlier studies of I. uriae (Olsen et al. 1995). For example, around Isla Magdalena bird species like Arctic tern spend their winter. Arctic terns have a transhemispheric migration route and could therefore act as reservoirs between north and south. So far we have not been able to genotype our positive samples. Of course it is most likely that they belong to the genospecies B. garinii but also B. burgdorferi sensu stricto and B. lusitaniae that are known to be involved in the marine cycle could be possible. If it would be another genospecies it would be a highly interesting result indicating that the transmission of pathogens between populations of I. uriae is larger than thought. In the study, DNA from Borrelia spirochetes was confirmed in 1.0 % (2 of 187) of the ticks. This prevalence is low compared to other studies. For example a recently conducted study from four islands on the northern hemisphere found an average prevalence of 26 % (Duneau et al. 2008). The low prevalence of Borrelia in this study can probably be an effect of that the ticks were divided before the analyses. Since the ticks were frozen for some months after they were divided this could have activated RNAses that break down the RNA. Also if the tick had a low number of spirochetes the result could be affected when the ticks were divided since one part could contain higher concentrations of spirochetes than the other half. During the cultivation no spirochetes at all were found in the samples. This was not surprising since the prevalence was so low. Often the success rate during cultivation of Borrelia spirochetes is low. There were also a lot of contaminating bacteria in the samples which could have competed the spirochetes.

4.3.2. Flavivirus When analysing the ticks from Isla Magdalena the prevalence for Flavivirus was 16 %. There are not many similar studies available to compare this result with but the when I. uriae from Macquarie Island was analysed the prevalence was 2 %. Since different techniques were used in these studies it is difficult to directly compare the results. In the study performed by Major et al. (2009) on Macquarie Island virus were first cultivated and then morphological identified to genus. After that primers were selected and used to amplify DNA. Probably there is a higher risk to miss positive ticks when this procedure is used. When the life stages were compared it was an increased prevalence for each stage even if it was not statistically significant. This indicates that the Flavivirus is transferred through a viremic host to the ticks and not transovarially from the female to the offspring. Flavivirus species often have limited geographical distribution, dependent on their hosts distribution (Gould et al. 2001). This offers opportunities for new strains to evolve and therefore studies on new locations are of interest. Since broad spectrum primers were used in this study there was a possibility to detect new Flavivirus. Therefore we had not excluded the possibility to find a new strain when the study was performed. During the Real-time PCR we were able to detect Flavivirus in the samples but unfortunately the sequencing failed. Since we prepared positive controls before the RNA-extraction which then were used during every step and gave positive results in the Real-time PCR we could confirm that the processes had been correctly performed. Therefore something else must have turned wrong. One reason to the problems could probably depend on the fact that the ticks were divided and then frozen for a longer period. This could have activated RNAses that breaks down the RNA. When melting peaks were analysed they were low compared to the positive controls. Since we have no idea which Flavivirus we are dealing with it is not easy to improve the procedure and use other primers. As mentioned it could be a totally new Flavivirus but more likely there are some of the already known that belong to the seabird group. As explained seabirds migrate long distances and thereby could act as reservoirs. If it had been possible to genotype the virus it had maybe been possible to draw conclusions about which birds that are involved in the transmission since the distribution for the Flavivirus species are so limited.

5. Acknowledgements First of all I want to thank Björn Olsen, Björn Hermann, Daniel Gonzales, Elsa Jourain, Jonas Bonnedahl and Jorge Hernandez who helped me to collect the ticks on Isla Magdalena and spent two very nice weeks together with me in Chile. Thanks also to Per- Eric Lindgren and everyone in his group in Linköping who gave me invaluable help during my lab work. I would also like to thank Sven Bergström and his group who teached me how to cultivate Borrelia spirochetes in Umea. Last but not least thanks to Jonas Waldenström who was my supervisor during this work.

6. References Barton, T. R., Harris, M. P., Wanless, S., (1995) Natural attachment duration of nymphs of the tick Ixodes uriae (Arcari: Ixodidae) on Kittiwake Rissa tridactyla nestlings. Experimental and applied acarology. Vol. 19: 499-509.

Benoit, J. B., Yoder, J. A., Lopez-Martinez, G., Elnitsky, M. A., Lee Jr, R. E., Denlinger, D. L., (2007). Habitat requirements of the seabird tick, Ixodes uriae (Acari: Ixodidae), from the Antarctic Peninsula in relation to water balance characteristics of eggs, nonfed and engorged stages. Journal of Comparative Physiology B Biochemical Systemic and Environmental Physiology. Vol. 177: 205–215.

Bergstrom, S., Haemig, P. D., Olsen, B., (1999). Distribution and abundance of the tick Ixodes uriae in a diverse subantarctic seabird community. International Journal for Parasitology. Vol. 85: 25–27.

Bergstrom, S., Haemig, P. D., Olsen, B., (1996). Increased mortality of black-browed albatross chicks at a colony heavily-infested with the tick Ixodes uriae. International Journal for Parasitology. Vol. 29: 1359–1361.

Chastel, C. E., (1988). Tick-borne virus infection of marine birds. In: Advances in disease vector research. Vol. 5 Springer-Verlag, New York, USA.

Davis, L. S., Darby, T. J., eds. (1990). Penguin biology. Academic press, United Kingdom.

Del Hoyo, J. D., Elliott, A., Sargatal, J., eds. (1992). Handbook of the birds of the world. Vol. 1. Ostrich to Ducks. Lynx Edicions, Barcelona, Spain.

Duneau, D., Boulinier, T., Gomez-Dıaz, E., Petersen, A., Tveraa, T., Barrett R. T., McCoy, K. D., (2008). Prevalence and diversity of Lyme borreliosis bacteria in marine birds. Infection, Genetics and Evolution Vol. 8: 352–359.

Eisen, L., Lane, R. S., (2002). Vectors of Borrelia burgdorferi sensu lato. In: Gray, J.S., Kahl, O., Lane, R.S., Stanek, G., (eds.), Lyme Borreliosis: Biology, Epidemiology and Control. CABI Publishing, New York, USA. 91–116.

Frenot, Y., Oliveira, E. D., Gauthier-Clerk, M., Deunff, J., Bellido, A., Vernon, P., (2001). Life cycle of the tick Ixodes uriae in penguin colonies: relationships with host breeding activity. International Journal for Parasitology. Vol. 31: 1040–1047.

Gauthier-Clerc, M., Clerquina, Y., Andrich, Y., (1998). Hyperinfestation by ticks Ixodes uriae: A possible cause of death in adult King Penguins, a long-lived seabird. Colonial Waterbirds Vol. 21: 229–233.

Clifford, C. M., (1979). Tick-borne virus infection of marine birds. InAdvances in disease vector research. Vol 5. Springer-Verlag, New York.

Gould, E. A., de Lamballerie, X., Zanotto, P. M. A., and Holmes, E. C., (2001). Evolution, Epidemiology and dispersal of flaviviruses revealed by molecular phylogenies. Advanced Virus Research. Vol. 57: 71–103.

Gylfe, Å., Bergström, S., Lundström, J., Olsen, B., (2000). Reactivation of Borrelia infection in birds. Nature Vol. 403: 724-725.

James, H., Oliver, JR., (1989). Biology and systematics of ticks (ACARI: IXODIDA). Annual Review of Ecology and Systematics. Vol. 20: 397–430

Ludwig, G. V., Iacono-Connar, L.C., (1993). Insect-transmitted vertebrate viruses: Flaviviridae. In Vitro Cellular and Developmental Biology. Vol. 29: 296–309.

Major, L., Linn, M. L., Slade, R. W., Schroder, W. A., Hyatt, A. D., Gardner, J., Cowley, J., Suhrbierm, A., (2009). Ticks Associated with Macquarie Island Penguins Carry Arboviruses from Four Genera. PLoS ONE Vol. 4: e4375.

Mangin, S., Gauthier-Clerc, M., Frenot, Y., Gendner, J-P., Maho, Y. L., (2003). Ticks Ixodes uriae and the breeding performance of a colonial seabird, king penguin Aptenodytes patagonicus. Journal of Avian Biology. Vol. 34: 30–34.

Mather, T. N., Ginsberg, S. H., (1994). Vector-host-pathogen relationships: Transmission dynamics of tick-borne infections. In: Sonenshine, D. E., Mather, T. N., (eds.), Ecological dynamics of tick-borne zoonoses. Oxford University Press. 68–90.

McCoy, K. D., Boulinier, T., Chardine, J. W., Danchin, E., Michalkis, Y., (1999). Dispersal and distribution of the tick Ixodes uriae within and among seabird host populations: The need for a population genetic approach. International Journal for Parasitology. Vol. 85: 196–202

McCoy, K. D., Chapuis, E., Tirard, C., Boulinier, T., Michalakis, Y., Le Bohec, C., Le Maho, Y., Gauthier-Clerc, M., (2005). Recurrent evolution of Host-Specialized Races in a Globally Distributed Parasite. Biological Sciences. Vol. 272: 2389–2395

McLean, R. G., Ubico, S. R., (2007) Arbovirus in birds. In: Thomas B. J., Hunter D. B., Atkinson C. T., (eds.), Infectious diseases of wild birds. Blackwell publishing 17–62.

Mehl, R., Traavik, T., (1983). The tick Ixodes uriae (Acaria:Ixodides) in seabird colonies in Norway. Fauna Norvegica Series B. Vol. 30: 94–107.

Moureau, G., Temmam, S., Gonzalez, J. P., Charrel, R. N., Grard, G., De Lamballerie, X., (2007). A real-time RT-PCR method for the universal detection and identification of flaviviruses. Vector-borne and zoonotic diseases. Vol. 7: 467–477.

Tälleklint, L., Jaenson, T. G. T., (1994) Transmission of Borrelia burgendorfi s. l. from mammal reservoirs to the primary vector of lyme borreliosis, Ixodes ricinus (Acari: Ixodidae), in Sweden. Journal of Medical Entomology. Vol. 31: 880–886.

Olsen, B., Jaenson, T. G. T., Noppa, L., Bunikis, J., Bergström S., (1993). A Lyme borreliosis cykle in seabirds and Ixodes uriae ticks. Nature Vol. 362: 340–342.

Olsen, B., Duffy, D. C., Jaenson, T. G. T., Gylfe, A., Bonnedahl, J., Bergstrom, S., (1995). Transhemispheric exchange of Lyme disease spirochetes by seabirds. Journal of Clinical Microbiology. Vol. 3: 3270–3274.

Olsen, B. (1995). Birds and Borrelia. Diss. Departments of microbiology and infectious diseases Umeå University, Umeå, Sweden. Solfjädern Offset AB, Sweden.

Olsen, B., (2007) Borrelia. In: Thomas B.J., Hunter D. B., Atkinson C. T., (eds.), Infectious diseases of wild birds. Blackwell publishing 341–351.

Sonenshine, D. E., (1991). Biology of ticks. New York, Oxford, Oxford University.

Warham, J., (1990) Introduction: The order Procellariiformes. In: Warham. J., (ed.). The petrels, their ecology and breeding systems. Academic Press, London, United Kingdom. 1–16.

Wilhelmsson, P., Fryland, L., Börjesson, S., Nordgren, J., Ekerfelt, K., Ernerud, J., Forsberg, P., Bergström, S., Lindgren, P-E., (2009). Characterization of Borrelia spp. in ticks detached from humans in Sweden. Manuscript.