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

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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 Degree project works made at the University of Kalmar, School of Pure and Applied Natural Sciences, can be ordered from: www.hik.se/student or University of Kalmar School of Pure and Applied Natural Sciences SE-391 82 KALMAR SWEDEN Phone + 46 480-44 73 00 Fax + 46 480-44 73 05 e-mail: [email protected] This is a degree project work and the student is responsible for the results and discussions in the report. 2 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. 3 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. 4 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 5 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). 6 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.
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