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Piratage de GTPases du trafic cellulaire humain par des régulateurs de pathogènes / Hijacking of cellular small GTPases by bacterial regulatorsFolly-Klan, Marcia 21 October 2014 (has links)
De nombreuses bactéries pathogènes piratent les machineries qui régulent le trafic cellulaire pour échapper à la réponse immunitaire de l’hôte ou faciliter leur multiplication. Les familles de petites protéines G Arf et Rab sont des régulateurs majeurs de la régulation du trafic intracellulaire et sont de ce fait des cibles privilégiées pour de nombreuses bactéries pathogènes. Ces pathogènes intracellulaires injectent dans leur cellule hôte des effecteurs capables d’imiter l’activité biochimique de leurs régulateurs pour activer, inhiber ou moduler l’activité des protéines. Ce projet porte sur l’étude de la famille de régulateurs bactériens RalF présents chez les bactéries Legionella pneumophila et Rickettsia prowazekii, les agents responsables de la maladie du Légionnaire et du typhus épidémique, respectivement. RalF possède un domaine catalytique apparenté à celui des facteurs d’échange eucaryotes dont le la fonction est d’activer la petite protéine G Arf. Cet effecteur bactérien agit comme un régulateur illégitime pour détourner les fonctions cellulaires de Arf1. L’objectif de cette thèse est de décrypter les mécanismes biophysiques, structuraux et dynamiques de régulation de la famille RalF pour comprendre les interactions protéine-protéine ou protéine- membrane impliqués dans le piratage de la fonction de Arf1. La combinaison d’études biochimiques et structurales nous ont permis de montrer que RalF est régulé par un cluster aromatique « senseur » de l’environnement lipidique lui permettant de localiser Arf1 à une membrane spécifique. / Many pathogenic bacteria highjack the cellular machineries that control cellular traffic in order to escape the host immune response, or to facilitate their replication. Small GTP-binding proteins (GTPases) of the Arf and Rab families, which are pivotal regulators in these processes, have thus been identified as targets for various human intracellular pathogens. To that purpose, these pathogens translocate bacterial effectors in the host cell, which mimics biochemical functions of their regulators to activate, inhibit or modulate of theses proteins. In this work, we study RalF, a family of bacterial regulators, which are translocated in host cells by the human intracellular pathogens Legionella pneumophila and Rickettsia prowazekii, which are responsible for Legionnaire’s disease and epidemic typhus, respectively. RalF possesses a catalytic domain related to eukaryotic exchange factors, which stimulate GDP/GTP exchange to activate small G-proteins Arf. This bacterial effector acts as an illegitimate regulator to divert cellular functions of Arf1. The aim of this work is to decipher biophysical, structural and dynamics regulation mechanisms of RalF family to understand the intramolecular protein-protein and protein-membrane interactions that underlie highjacking of Arf1 function. Using biochemical and structural analyses we showes how RalF is regulated by a membrane “sensor” to recruit Arf1 at specific membrane locations.
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Investigation of Anaplasma phagocytophilum and Anaplasma marginale adhesin-host cell interactionsHebert, Kathryn S. 01 January 2016 (has links)
Anaplasma phagocytophilum and A. marginale are the etiologic agents of bovine anaplasmosis and human granulocytic anaplasmosis, respectively. As obligate intracellular pathogens, binding and entry of host cells is a prerequisite for survival. The molecular events associated with these processes are poorly understood. Identifying the adhesins mediating binding, delineating their key functional domains, and determining the molecular determinants to which they bind not only benefits better understanding of Anaplasma spp. pathobiology, but could also benefit the development of novel approaches for protecting against infection. We previously demonstrated that A. phagocytophilum outer membrane protein A (ApOmpA) is critical for bacterial binding and entry host through recognition of α2,3-sialic acid and α1,3-fucose of its receptors, including 6-sulfo-sLex. In this study, we determined that two amino acids, G61 and K64, within its binding domain (ApOmpA59-74), are essential for ApOmpA function. We also confirmed the ability of ApOmpA to act as an adhesin and invasin as it conferred adhesiveness and invasiveness to inert beads. We next extended our studies to A. marginale as it also expresses OmpA (AmOmpA) and its role in infection has not been studied. Molecular models of ApOmpA and AmOmpA were nearly identical, especially in the ApOmpA binding domain and its counterpart in AmOmpA. Antisera raised against AmOmpA or its putative binding domain inhibit A. marginale infection. AmOmpA G55 and K58 are contributory and K59 is essential for AmOmpA to bind to host cells. AmOmpA binding is dependent on α2,3-sialic acid and α1,3-fucose. Coating inert beads with AmOmpA conferred the ability to bind to and be taken up by host cells, confirming that it acts as an adhesin and invasin. 6-sulfo-sLex is dispensable for AmOmpA binding and A. marginale infection. ApOmpA works cooperatively with Asp14 (14-kDa A. phagocytophilum surface protein) to promote optimal infection of host cells. We found that Asp14 is conserved across A. phagocytophilum strains and in A. marginale and confirmed the ability of Asp14 to act as an adhesin and invasin as it conferred adhesiveness and invasiveness to inert beads. Collectively, this work advances our understanding of A. phagocytophilum and A. marginale adhesion and invasion of host cells.
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Identification des arthropodes vecteurs et des micro-organismes associés par MALDI-TOF-MS / Identification of arthropods vectors and associated micro-organisms by MALDI-TOF MSYssouf, Amina 06 October 2014 (has links)
Les arthropodes vecteurs sont hématophages et peuvent assurer la transmission biologique active d'un agent pathogène responsable de maladies humaines ou animales. La lutte anti-vectorielle et la surveillance épidémiologique des vecteurs sont essentielles dans la stratégie de lutte contre les maladies vectorielles. Disposer d'outils d'identification précis, fiable et rapides des vecteurs et des pathogènes associés est indispensable. Ainsi dans ce projet nous avons évalué l'utilisation du MALDI-TOF MS pour identifier les arthropodes vecteurs ainsi que la détection des pathogènes associés. La première partie de notre travail consistait à utiliser MALDI TOF pour identifier les tiques, moustiques et les puces. Nous avons déterminé quelle partie du spécimen permettait d'obtenir une reproductibilité des spectres et une identification correcte par des tests à l'aveugle après création d'une base de données de référence. La deuxième partie consistait à utiliser le MALDI-TOF MS pour détecter des Rcikettsies associés aux tiques dont Rickettsia conorii et R. slovaca, deux pathogènes humains transmis respectivement par Rhipicephalus sanguineus et Dermacentor marginatus. Des variations spectrales étaient obtenues entre les spécimens infectés et non infectés, avec des masses spécifiques liés à l'infection des tiques par les rickettsies. La technique d'identification était validée par des tests à l'aveugle. Les résultats obtenus permettent de conclure que le MALDI TOF pourra être utilisé dans l'avenir pour identifier les tiques prélevées chez des patients, les arthropodes vecteurs lors des enquêtes entomologiques et préciser la prévalence d'infection de ces arthropodes. / Arthropods are vectors bloodsucking and can ensure the active biological transmission of a pathogen responsible of human or veterinary diseases. The vector control and vectors epidemiological surveillance are essential in the strategy against the vectors-borne diseases. Accurate, reliable and rapid identification of vectors and associated pathogens are essential. Thus, in this project we evaluated the use of MALDI-TOF MS for the arthropods vectors identification as well as for the detection of associated pathogens. This proteomics technology emerged since few years ago and is currently used in routine for bacteria identification in many microbiology laboratories. In the first part of our work, we used the MALDI TOF to identify the tick, mosquito and flea species. For each arthropod, we determined which part allowed obtaining reproducible spectra by MALDI TOF and correct identification by blind test, after reference database creation. The second part consisted to use the MALDI-TOF MS to detect the associated Rickettsia in ticks including Rickettsia conorii and R. slovaca, two human pathogens transmitted by Rhipicephalus sanguineus and respectively Dermacentors marginatus. The spectral variations were obtained between infected and non infected specimens with specific masses related to the tick infection by Rickettsia. The identification technique of not or infected ticks was validated by blind tests. The obtained results allowed concluding that the MALDI-TOF MS could be used in the future to identify the ticks removed from patient, the arthropods vectors and during entomological survey and determine the prevalence of infection of these arthropods.
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Identification of the tick-borne pathogens Anaplasma phagocytophilum, Neoehrlichia mikurensis and Rickettsia in Swedish ticks : Investigation of transovarial transmission and co-infectionJönsson, Johanna January 2016 (has links)
Globally, vector borne diseases cause more than a million deaths each year and more than a billion infections in humans. Ticks are of big medicinal importance since they can transmit pathogens that can cause serious infections. Some recently discovered pathogens that can cause infections in humans are Anaplasma phagocytophilum (A. phagocytophilum) that can cause human granulocytic anaplasmosis (HGA) and Candidatus Neoehrlichia mikurensis (N. mikurensis) that can cause Neoehrlichiosis. It is still widely unknown how prevalent these pathogens are, if ticks can be infected with both of these pathogens and if these pathogens can be transovarially transmitted from adult female to egg and larvae. This study aims to screen for these pathogens in collected ticks from southern Sweden and to detect eventual co-infections and transovarial transmission. A real-time qPCR assay targeting the 16S rRNA gene of N. mikurensis and other Anaplasmataceae was applied on 1356 Ixodes ricinus (I. ricinus) ticks collected from 5 sites in southern Sweden. Positive samples were subjected to Sanger sequencing. A. phagocytophilum occurred in 4.64 % of the ticks, N. mikurensis occurred in 1.33 % of the ticks and also Rickettsia was found to occur in 6.27 % of the ticks. No co-infection was detected. Some samples of tick larvae showed positive results after qPCR, indicating transovarial transmission, but none of the sequences were readable.
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Epidemiological and Bacteriological Aspects of Spotted Fever Rickettsioses in Humans, Vectors and Mammals in SwedenElfving, Karin January 2013 (has links)
Rickettsiae are obligate intracellular gram-negative bacteria transmitted by arthropod vectors. Rickettsiae sometimes cause disease in humans, typically with high fever, headache and occasionally an eschar. In Sweden, Rickettsia helvetica, belonging to the spotted fever group, is the only tick-transmitted rickettsia found free in nature. The pathogenic roll of R. helvetica has not been fully investigated, but it has been implicated in aneruptive fever and cardiac disease. This thesis describes parts of the transmission pathways of rickettsiae in Sweden. Rickettsia infection rates in ticks collected from birds were analysed, and the birds’ role as disseminators and reservoirs was studied. We found that more than one in ten ticks was infected with rickettsia bacteria, predominantly R. helvetica, and that migrating birds contribute not only to long-distance dispersion of bacteria, but also to an inflow of novel and potentially pathogenic rickettsia species, in this case R. monacensis and R. sp. strain Davousti-like species, into Sweden. Further, wild and domestic animals were found to have seroreactivity against R. helvetica, which shows that they are exposed and susceptible to rickettsia. Their role as reservoirs has not been determined, yet they may indirectly be involved in transmission of rickettsia to humans by infected ticks feeding on them. The seroreactivity in humans was also studied. Patients investigated for suspected Borrelioses and blood donors had detectable antibodies against Rickettsia spp., with the highest prevalence detected in the suspected Borreliosis group. This shows that humans in Sweden are exposed to and develop an immune response against rickettsia. The suspicion that R. helvetica may cause severe symptoms was verified by a patient with subacute meningitis where the bacterium was shown for the first time to cause an invasive infection with CNS involvement and where the bacterium was isolated from the patient’s cerebrospinal fluid. Growth characteristics and morphology of R. helvetica were studied to better understand invasiveness and virulence. The findings indicate that the invasiveness is comparable with other rickettsia, though R. helvetica seems to have a stable but slightly slower growth. Rickettsia helvetica is endemic in Sweden and therefore needs to be considered when investigating disease after a tick bite. / Rickettsia är en liten, strikt intracellulär, gramnegativ bakterie som sprids med vektorer som fästingar, löss och loppor. Bakterien kan orsaka Rickettsios hos människa, en sjukdom där de vanligaste symtomen är hög feber, huvudvärk, muskelvärk och i vissa fall ett bettmärke (eschar). I Sverige är Rickettsia helvetica, som tillhör spotted fever gruppen (SFG), den enda fästingöverförda rickettsia bakterien som hittats allmänt i naturen. Patogeniciteten för R. helvetica är ofullständigt utredd, men ”aneruptive fever” och hjärtmuskelinflammation har rapporterats. Avhandlingen beskriver delar av smittkedjan för SFG rickettsia i Sverige. Bakteriernas förekomst i fästingar plockade från fåglar har studerats, likaså det ekologiska tryck som flyttfåglars bärarskap av infekterade fästingar bidrar med när de korsar olika världsdelar. Mer än var tionde fästing var infekterad med rickettsia bakterier, i huvudsak R. helvetica. Det visade sig att flyttfåglar bidrar inte bara till långväga spridning av bakterier utan även till införsel av nya potentiellt patogena rickettsiaarter, i detta fall identifierades R. monacensis och en R. sp strain Davousti liknande art. Vidare analyserades seroreaktivitet mot Rickettsia helvetica hos både tamdjur och vilda djur, vilket visade på antikroppsutveckling, som uttryck för smittexposition, i mer än vart femte djur. Djurens roll som reservoar för bakterien är inte klarlagd, men oavsett är djuren indirekt involverade i spridningen av bakterien till människa via infekterade fästingar som suger blod. Seroreaktivitet hos människa har också studerats. Patienter, provtagna på grund av misstanke om borreliainfektion, samt blodgivare hade detekterbara antikroppar mot Rickettsiae, med högst prevalens i gruppen med misstänkt borreliainfektion. Fynden visar att människor i Sverige är exponerade för och utvecklar en immunreaktion mot rickettsia. Att R. helvetica skulle kunna ge allvarlig sjukdom verifieras av ett patientfall med subakut meningit där bakterien för första gången visats ge invasiv infektion med påverkan på nervsystemet (CNS engagemang) och där bakterien isolerats från patientens ryggmärgsvätska. Morfologi och tillväxtegenskaper för R. helvetica undersöktes för att bättre förstå bakteriens invasivitet och virulens. Fynden indikerar att invasiviteten är jämförbar med andra rickettsiaarter men R. helvetica verkar ha en stabil men något långsammare tillväxt. Rickettsia helvetica är endemisk i Sverige och måste tas i beaktande vid sjukdomsutredning efter ett fästingbett.
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The Microbial Associates and Putative Venoms of Seed Chalcid Wasps (Hymenoptera: Torymidae: Megastigmus)Paulson, Amber Rose 20 December 2013 (has links)
Conifer seed-infesting chalcids of the genus Megastigmus (Hymenoptera: Torymidae) are important forest pests. At least one species, M. spermotrophus Wachtl, has been shown to be able to manipulate the seed development of its host, Douglas-fir (Pseudotsuga menziesii) in remarkable ways, such as redirecting unfertilized ovules that would normally abort. The mechanism of host manipulation is currently unknown. Microbial associates and venoms are two potential mechanisms of host manipulation. Microbial associates are emerging as an important player in insect-plant interactions. There is also evidence that venoms may be important in gall-induction by phytophagous wasps. PCR and 16S rRNA pyrosequencing was used to characterize the microbial associates of Megastigmus and transcriptomic sequencing was used to identify putative venoms that were highly expressed in female M. spermotrophus. The common inherited bacterial symbionts Wolbachia and Rickettsia were found to be prevalent among several populations of Megastigmus spp. screened using a targeted PCR approach. A member of the Betaproteobacteria, Ralstonia, was identified as the dominant microbial associate of M. spermotrophus using 16S rRNA pyrosequencing. The transcriptome of M. spermotrophus was assembled de novo and three putative venoms were identified as highly expressed in females. One of these putative venoms, Aspartylglucosaminidase, (AGA) appears to have originated through gene duplication within the Hymenoptera and has been identified as a major venom component of two divergent parasitoid wasps. AGA was identified as a promising candidate for further investigation as a potential mechanism of early host manipulation by M. spermotrophus. / Graduate / 0353 / 0410 / 0715 / apaulson@shaw.ca
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Jämförelse av Fluidigm-PCR och realtids-PCR vid detektion av Rickettsia spp. : Samt undersökning av risken att drabbas av infektion efter bett av rickettsiainfekterad fästing / Comparison between Fluidigm-PCR and real-time PCR for detection of Rickettsia spp. : And evaluation of the risk of getting an infection after being bitten by a tick infected with Rickettsia spp.Estberg, Evelina, Dulic, Mirela January 2018 (has links)
Fästingburna infektioner är ett ökande problem, och därmed även infektioner orsakade av Rickettsia spp. Syftet med studien var att undersöka risken att drabbas av en infektion efter bett av rickettsiainfekterad fästing. Specificitet och sensitivitet av Fluidigm-PCR jämfördes mot real time polymerase chain reaction (realtids-PCR) vid detektion av Rickettsia spp. i fästingar som bitit människor. Vidare undersöktes om det finns någon korrelation mellan fästingens blodsugningstid och serokonversion mot Rickettsia spp. 753 fästingar lämnades in av 104 deltagare i Sverige och på Åland. Fästingarna analyserades med realtids-PCR för att detektera gltA-genen som är specifik för Rickettsia spp. 3,5 % av fästingarna var positiva för Rickettsia spp. med realtids-PCR. Vid analysering med Fluidigm-PCR av samma material blev 1,3 % av proverna positiva. Beräkningar som gjordes med realtids-PCR som referens visade att Fluidigm-PCR har sämre specificitet och sensitivitet jämfört med realtids-PCR. Deltagare som serokonverterade (n=5) lämnade endast in rickettsianegativa fästingar som därför inte kunde kopplas till infektionen. Därmed kunde inga slutsatser dras om risken att drabbas av en infektion efter bett av rickettsiainfekterad fästing eller om det föreligger någon korrelation mellan fästingens blodsugningstid och serokonversion. / Tick-borne infections are increasing, including infections caused by Rickettsia spp. The aim of this study was to examine the risk of developing an infection after being bitten by a tick infected with Rickettsia spp. Specificity and sensitivity of a Fluidigm-PCR assay were compared to real time polymerase chain reaction (real-time PCR) assay when detecting Rickettsia spp. in ticks that had bitten humans. Possible correlation between the tick's feeding time and seroconversion against Rickettsia spp. was also investigated. A total of 753 ticks from 104 participants in Sweden and the Åland Islands (Finland) were analyzed with real-time PCR to detect the gltA gene specific for Rickettsia spp. 3.5 % of the samples were positive for Rickettsia spp. with real-time PCR, while only 1.3 % of the samples were positive with Fluidigm-PCR. Calculations showed that Fluidigm-PCR assay has lower specificity and sensitivity than the real-time PCR assay. Unfortunately, no conclusions could be drawn considering correlation between the tick's feeding time and seroconversion of the bitten humans since no participants who had seroconverted had also submitted ticks containing Rickettsia spp. Therefore, no conclusions could be drawn considering the risk of developing an infection after being bitten by a tick infected with Rickettsia spp. / STING-studien
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Fauna, sazonalidade e riquetsias de carrapatos em área do Cerrado goianoMartins, Maria Marlene 17 August 2016 (has links)
Conselho Nacional de Desenvolvimento Científico e Tecnológico / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Visando contribuir com a descrição da diversidade e ecologia de carrapatos e riquétsias no Cerrado brasileiro, este estudo foi conduzido em uma fazenda particular e suas imediações em Araguapaz, Goiás. O trabalho foi realizado durante quatro anos em 11 coletas, sendo oito em estações consecutivas em três fitofisionomias distintas. Coletou-se carrapatos do ambiente, de animais domésticos, aves e pequenos mamíferos silvestres e, em uma amostra destes, procurou-se por DNA de riquétsias. No ambiente foram coletados Amblyomma sculptum, A. ovale, A. parvum, A. naponense, A. rotundatum e Ornithodoros sp. O ambiente com infestação mais intensa e diversa foi a Mata de Galeria e o carrapato A. sculptum exibiu uma distribuição sazonal bem definida com larvas e ninfas prevalecendo nas estações mais secas e adultos nas mais úmidas. Em cães foram encontrados A. sculptum, A. ovale, A. parvum e Rhipicephalus sanguineus; em equídeos A. sculptum, Dermacentor nitens e R.(Boophilus) microplus; em bovinos R. (Boophilus) microplus e A. sculptum. Nas aves silvestres foram coletadas ninfas de A. sculptum e A. nodosum. Nos pequenos roedores coletou ninfas de A. parvum, larvas de Amblyomma spp e larvas de Ornithodoros sp. Coletas fortuitas em répteis, resultaram em ninfas e adultos de A. rotundatum e larvas de Amblyomma sp. Duas espécies de riquetsias do grupo da febre maculosa foram identificadas R. amblyommii e candidatus R. andeanae ambas em A. parvum e R. bellii em A. rotundatum. O carrapato Ornithodoros sp encontrado diverge morfologicamente e geneticamente das espécies conhecidas constituindo-se em uma nova espécie. De forma global ficou evidente que os animais na área estudada são expostos a uma diversidade maior de macroparasitos (carrapatos) e microparasitos (bactérias) do que em áreas muito mais antropizadas. O papel desta diversidade maior no desenvolvimento de doenças, na prevenção destas, merece investigações adicionais. / Aiming contributing to the description of species and of the ecology of ticks and Rickettsia in the Cerrado (savannah) this research was conducted in private farm and surroundings in Araguapaz county, Goiás. Overall eleven samplings, eight in consecutive seasons in three phytofisiognomies, were done along four years. Ticks were collected from the environment, domestic animals, birds and small mammals and Rickettsia DNA was searched for in a sample of this ectoparasites. Amblyomma sculptum, A. ovale, A. parvum, A. naponense, A. rotundatum and Ornithodoros sp. were collected from the environment and gallery forests exhibited the overall highest infestation and tick species diversity. A. sculptum ticks exhibited a seasonal pattern with larvae and nymphs prevailing in the driest seasons and adults in most humid ones. Dogs were parasitized by A. sculptum, A. ovale, A. parvum and Rhipicephalus sanguineus; horses by A. sculptum, Dermacentor nitens and Rhipicephalus (Boophilus) microplus; cattle by Rhipicephalus (Boophilus) microplus, and A. sculptum. A. sculptum and A. nodosum nymphs were collected from birds and A. parvum nymphs, Amblyomma sp. and Ornithodoros sp. larvae from small mammals. Unplanned tick collections from reptiles yielded A. rotundatum nymphs and adults as well as Amblyomma sp. larvae. Two spotted fever Rickettsia species were identified; R. amblyommii and candidatus R. andeanae both in A. parvum as well as R. bellii in A. rotundatum. The Ornithodoros sp. tick found diverge both morphologically and genetically from known species and a new tick species may be supposed. Broadly it is evident that in the studied area animals are exposed to a higher macroparasite (ticks) and microparasites (bactéria) that those from more anthroposized areas. The role of such higher parasite diversity in the development or prevention of diseases should be further investigated. / Tese (Doutorado)
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Einfluss von Umwelt- und individuellen Faktoren auf die Prävalenz von Rickettsia spp. in Kleinsäugern und ZeckenArz, Charlotte 10 June 2024 (has links)
Die am häufigsten vorkommenden Zeckenarten in Deutschland sind der gemeine Holzbock (Ixodes ricinus) und die Auwaldzecke (Dermacentor reticulatus). Für beide Arten stellen Kleinsäuger Wirte für die subadulten Stadien dar und bestimmen so ihr gemeinsames Vorkommen in den Habitaten der Wirtstiere. Zecken fungieren als Vektoren und/oder Reservoire für verschiedene Pathogene und sind die Vektoren von fast allen Rickettsienarten der Zeckenstichfiebergruppe. Die in Deutschland vorkommenden Rickettsienarten Rickettsia helvetica und Rickettsia raoultii wurden mit Erkrankungen des Menschen in Verbindung gebracht.
Untersuchungen über den Einfluss von umweltassoziierten und individuellen Faktoren auf die Rickettsienprävalenz in Zecken und Kleinsäugern sind rar. Aufgrund dessen war es Ziel dieser Studie, die Prävalenz von Rickettsien in Zecken und Kleinsäugern nicht nur in Bezug auf Spezies und Saison zu untersuchen, sondern auch verschiedene Habitatstrukturen zu betrachten. Zusätzlich wurden Daten zu Ko-Infektionen der beiden Bakteriengattungen Rickettsien und Borrelien in Zecken und Kleinsäugern erhoben.
Untersucht wurden den Hainich-Dün Nationalpark in Thüringen umschließende Gebiete. Insgesamt standen DNA-Proben von 1167 Kleinsäugerohrsegmenten (zehn Spezies; Frühjahr und Sommer 2017-19) und 1098 Zecken (zwei Spezies; Frühjahr, Sommer, und Herbst 2018 und 2019) zur Verfügung. Die Untersuchungsstellen wurden dabei für Kleinsäuger in die Habitate Waldgebiet und Grasland und für Zecken in die Habitate Waldgebiet und angrenzender Ökoton zwischen Waldgebiet und Grasland eingeteilt. Die DNA-Proben wurden mittels einer quantitativen real-time PCR auf das Vorhandensein des gltA Gens von Rickettsien untersucht. Positive Proben wurden mithilfe einer das ompB Gen detektierenden konventionellen PCR auf Rickettsia-Speziesebene durch anschließende Sanger Sequenzierung charakterisiert. Die statistische Auswertung umfasste unter Anderem die Errechnung von Konfidenzintervallen (95 % KI) und die Erstellung mehrerer linear gemischter generalisierter Modelle (GLMM).
Rickettsien-DNA wurde in 11,4 % (n=125) der Zecken gefunden. Innerhalb der I. ricinus Zecken (8,6 %; 88/1018) war die Rickettsienprävalenz niedriger als in D. reticulatus Zecken (46,3 %; 37/80) (GLMM: p=<0,001). Im Herbst gesammelte Zecken waren signifikant häufiger Rickettsia-positiv als Zecken aus dem Frühjahr und Sommer (GLMM: jeweils p=<0,001). Das Habitat hatte keinen Einfluss auf die Infektionswahrscheinlichkeit der Zecken (GLMM: p=0,7109). Adulte Zecken waren signifikant häufiger Rickettsia-positiv als Nymphen (GLMM: p=0,0199). Es wurden 87 Rickettsia-positive Zeckenproben auf Spezieslevel bestimmt. Alle 75 I. ricinus Zecken konnten als R. helvetica und alle 12 D. reticulatus Zecken konnten als R. raoultii bestimmt werden.
In 9,9 % (n=115) der Kleinsäuger (8/10 Arten: Microtus arvalis, Microtus agrestis, Clethrionomys glareolus, Apodemus flavicollis, Apodemus sylvaticus, Apodemus agrarius, Sorex araneus, Sorex minutus) konnte Rickettsien-DNA detektiert werden. Tiere aus den Waldgebieten waren häufiger Rickettsia-positiv als Tiere aus dem Grasland (GLMM: p=<0,001). Der Faktor Saison hatte keinen Einfluss auf die Infektionswahrscheinlichkeit (GLMM: p=0,1062). Die Rickettsienspezies konnte ausschließlich in Proben zweier A. flavicollis als R. helvetica identifiziert werden.
Die Zahlen der Ko-infektionen waren bei Zecken (0,8 %) und Kleinsäugern (0,8 %) gering. Lediglich S. araneus zeigte eine vergleichsweise hohe Ko-Infektionsrate (10,0 %; 2/20).
Die detektierte Rickettsienprävalenz in Kleinsäugern und Zecken deckt sich jeweils mit den Prävalenzen voriger Studien aus Deutschland. Interessanterweise beeinflusste das Habitat (höher im Wald als im Grasland) ausschließlich die Prävalenz innerhalb der untersuchten Kleinsäugerpopulation, nicht jedoch die Prävalenz innerhalb der untersuchten Zecken. Eine mögliche Erklärung hierfür könnte die hohe Prävalenz der vermehrt im Wald gefangenen Arten A. flavicollis und A. sylvaticus sein. Rickettsia helvetica und R. raoultii wurden jeweils ausschließlich in ihren assoziierten Zeckenspezies detektiert.:Inhaltsverzeichnis
1 EINLEITUNG 1
2 LITERATURÜBERSICHT 3
2.1 ZECKEN 3
2.1.1 Taxonomie der Zecken 3
2.1.2 Habitat der Zecken 3
2.1.3 Lebenszyklus, Blutmahlzeiten, Wirte 4
2.1.4 Transmissionswege von Pathogenen innerhalb einer Zeckenpopulation 5
2.1.5 Vektorfunktion von Ixodes ricinus 6
2.1.6 Vektorfunktion von Dermacentor reticulatus 7
2.2 KLEINSÄUGER 8
2.2.1 Taxonomie 8
2.2.2 Wühlmäuse der Gattung Microtus 9
2.2.3 Die Wühlmäuse Clethrionomys glareolus und Arvicola amphibius 10
2.2.4 Echte Mäuse der Gattung Apodemus 11
2.2.5 Spitzmäuse: Sorex spp. und Crocidura russula 12
2.3 RICKETTSIA SPP. 13
2.3.1 Taxonomie und Aufbau des Bakteriums 13
2.3.2 Vektoren von Rickettsien der Zeckenstichfieber-Gruppe (SFG) und Übertragung auf den Wirt 14
2.3.3 Verbreitung von Rickettsien der Zeckenstichfieber-Gruppe in Deutschland, Europa, Welt 15
3 PUBLIKATION 19
4 DISKUSSION 38
5 ZUSAMMENFASSUNG 48
6 SUMMARY 50
7 ANHANG/REFERENZEN 52
7.1 LITERATURVERZEICHNIS 52
7.2 ABBILDUNGSVERZEICHNIS 62
7.3 TABELLENVERZEICHNIS 62
8 DANKSAGUNG 63
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The Origin of the Genus Flavivirus and the Ecology of Tick-Borne PathogensPettersson, John H.-O. January 2013 (has links)
The present thesis examines questions related to the temporal origin of the Flavivirus genus and the ecology of tick-borne pathogens. In the first study, we date the origin and divergence time of the Flavivirus genus. It has been argued that the first flaviviruses originated after the last glacial maximum. This has been contradicted by recent analyses estimating that the tick-borne flaviviruses emerged at least before 16,000 years ago. It has also been argued that the Powassan virus was introduced into North America at the time between the opening and splitting of the Beringian land bridge. Supported by tip date and biogeographical calibration, our results suggest that this genus originated circa 120,000 (156,100–322,700) years ago if the Tamana bat virus is included in the genus, or circa 85,000 (63,700–109,600) years ago excluding the Tamana bat virus. In the second study we estimate the prevalence of tick-borne encephalitis virus (TBEV) in host-seeking Ixodes ricinus from 29 localities in Sweden and compare our data with those of neighbouring countries. Nymphs and adult ticks were screened for TBEV using a real-time PCR assay. The mean TBEV prevalence for all tick stages combined was 0.26% for Sweden and 0.28% for all Scandinavian countries, excluding Iceland. The low prevalence of TBEV in nature may partly be explained by the fact that TBEV occurs in spatially small foci and that the inclusion of ticks from non-infected foci will reduce the prevalence estimate. In the third and fourth study, we conducted the first large-scale investigations to estimate the prevalence and geographical distribution of Anaplasma spp. and Rickettsia spp. in host-seeking larvae, nymphs and adults of I. ricinus ticks in Sweden. Ticks were collected from several localities in central and southern Sweden and were subsequently screened for the presence of Anaplasma spp. and Rickettsia spp. using a real-time PCR assay. For all active tick stages combined, the mean prevalence of Anaplasma spp. and Rickettsia spp. in I. ricinus in Sweden was estimated to 1.1% and 4.8%, respectively. It was also shown that A. phagocytophilum and R. helvetica are the main Anaplasma and Rickettsia species occurring in Sweden.
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