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Borne Bacteria from Ethiopia AIX-MARSEILLE UNIVERSITE FACULTE DE MEDECINE DE MARSEILLE ECOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTE Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes « URMITE ». THESE DE DOCTORAT présentée par Bersissa Kumsa MOLECULAR INVESTIGATION OF ARTHROPODS AND VECTOR- BORNE BACTERIA FROM ETHIOPIA Soutenue le 02 Décembre 2014 pour obtenir le grade de Docteur d’Université d’Aix-Marseille Spécialité : Maladies Infectieuses et Pathologies Humaines Membres du Jury : Prof. Philippe Parola (MD, PhD) Directeur de thèse Prof. Sarah Bonnet (MD, PhD) Rapporteur Prof. Pierre Marty (MD, PhD) Rapporteur Prof. Pierre-Edouard Fournier (MD, PhD) Examinateur Laboratoire d’accueil : Unité de Recherche sur les Maladies Infectieuses et Tropicales émergentes « URMITE » UM63, CNRS7278, IRD 498, Inserm 1095, Faculté de Médecine Avant Propos Le format de présentation de cette thèse correspond à une recommandation de la spécialité Maladies Infectieuses et Microbiologie, à l‟intérieur du Master de Sciences de la Vie et de la Santé qui dépend de l‟Ecole Doctorale des Sciences de la Vie de Marseille. Le candidat est amené à respecter des règles qui lui sont imposées et qui comportent un format de thèse utilisé dans le Nord de l‟Europe permettant un meilleur rangement que les thèses traditionnelles. Par ailleurs, la partie introduction et bibliographie est remplacée par une revue envoyée dans un journal afin de permettre une évaluation extérieure de la qualité de la revue et de permettre à l‟étudiant de le commencer le plus tôt possible une bibliographie exhaustive sur le domaine de cette thèse. Par ailleurs, la thèse est présentée sur article publié, accepté ou soumis associé d‟un bref commentaire donnant le sens général du travail. Cette forme de présentation a paru plus en adéquation avec les exigences de la compétition internationale et permet de se concentrer sur des travaux qui bénéficieront d‟une diffusion internationale. Professeur Didier RAOULT 1 2 Acknowledgements First and foremost I would like to express my sincere gratitude to my PhD supervisor, Prof. Didier Raoult, the Director of the laboratory and renowned scientist of Clinical microbiology, for his continuous guidance, suggestions and constructive comments throughout my research. I also would like to thank him very much for giving me a very important chance to do a PhD degree, for his financial support (AP+HM) of my PhD research at his laboratory. I am very pleased and thankful to my PhD supervisor Prof. Philippe Parola for his constant suggestions, lots of good ideas, rigorous guidance, very professional comments, great advice and mentor for my PhD research. I am greatly thankful to my 3rd PhD supervisor Dr. Cristina Scolvoccshi for her constant suggestions, lots of critically important comments and great advice during my PhD research. I would like to thank the reviewers of my PhD thesis Prof. Sarah Bonnet and Prof Marthy Pierre for their high quality professional advices and detailed review during the preparation of the thesis. Their sincere suggestions were critically important to improve the quality of this PhD thesis. 3 4 My heartfelt thanks go to the technicins of URMITE laboratory particularily Annick Bernard, Laurence Thomas, Veronique Brice, Stephanie Junoy, Marielle Bedotto, Anne-Marie Gottrau and Jean-Michel Berenger for their technical support during the whole of my PhD research in the Laboratory. Also my deepest heartfelt thanks go to the secretaries of URMITE especially MS. Francine Simula and Valerie Filosa for their very kind and honest help regarding very important issues like accommodation and VISA arrangements throughout my 3 years stay in Marseille for PhD research at the laboratory. I take this opportunity to thank my wife Tadelech Fite and son Barecha Bersissa for their great love, moral support and patience throughout my long absence from the lovely home. Lastly but not least my great thanks goes to my beloved mother Dinkitu Abetu Morka, for her moral support, great love and thinking about me for the entire of her life. 5 TABLE OF CONTENTS Acknowledgements 3 INTRODUCTION 13 2. REVIEW 19 2.1. Article 1 21 Negelected Vector-borne Bacterial Zoonoses in East Africa. Kumsa, B., Raoult, D., Parola, P., To be submitted to Acta Tropica 3. TICKS 121 3.1. Article 2 127 Spotted fever group rickettsiae in ixodid ticks in Oromia, Ethiopia. Kumsa, B., Socolovschi, C., Raoult, D., Parola, P., 2014. Ticks Tick Borne Dis 3.2. Article 3 137 Occurrence and genotyping of Coxiella burnetii in ixodid ticks in Oromia, Ethiopia. Kumsa, B., Socolovschi, C., Almeras, L., Raoult, D., Parola, P., To be submitted to Plos Neglected tropical Diseases. 3.3. Article 4 165 New Borrelia species detected in ixodid ticks in Oromia, Ethiopia. Kumsa, B., Socolovschi, C., Raoult, R., Parola, P., To be submitted to Ticks Tick Borne Dis. 3.4. Article 5 193 Morphological, MALDI TOF Mass spectrometry and molecular identification of ixodid tick species in Oromia, Ethiopia. Kumsa, B., Almeras, L., Yussuf, A., Mediannikov, O., Raoult, D., Parola, P., To be submitted to Veterinary Parasitollogy. 6 4. FLEAS 237 4.1. Article 6 215 Molecular detection of Rickettsia felis and Bartonella henselae in dog and cat fleas in central Oromia, Ethiopia. Kumsa, B., Parola, P., Raoult, D., Socolovschi, C., Am. J. Trop. Med. Hyg., 90(3), 2014, pp. 457–462 5. LICE AND SHEEP KED 249 5.1. Article 7 227 Molecular detection of Acinetobacter species in the lice and keds of domestic animals in Oromia Regional State, Ethiopia. Kumsa, B., Socolovschi, C., Raoult, D., Parola, P., PLoS ONE 7(12): e52377. doi:10.1371/journal.pone.0052377. 5.2. Article 8 241 Bartonella melophagi in Melophagus ovinus (sheep ked) collected from sheep in northern Oromia, Ethiopia. Kumsa, B., Raoult, D., Parola, P., Socolovschi, C., Comparative Immunology, Microbiology and Infectious Diseases 37 (2014) 69– 76. 6. CONCLUSIONS 249 6.1. Conclusions and perspectives 251 Bibliography 281 7 Résumé Les arthropodes comme les tiques, les puces, les poux et les moutons ked se trouvent dans toutes les régions du monde et sont plus fréquents dans les pays tropicaux. Les vecteurs arthropodes hématophages comme les tiques, les puces, les poux et les moutons ked transmettent les bactéries qui sont généralement associés aux maladies fébriles chez l'homme. Les informations sur les bactéries transmises par les arthropodes est rare en Ethiopie. Ainsi, l'objectif principal de cette thèse est d'accroître les connaissances sur les bactéries à transmission vectorielle en Ethiopie par l'exploration de leur présence dans plusieurs types d'arthropodes y compris les tiques, les puces, les poux et les moutons ked prélevés sur des animaux dans plusieurs départements du pays. En outre, nous avons fait une expérience sur les nouveaux outils pour identifier les tiques par MALDI-TOF MS protéines profilage et des méthodes moléculaires. Notre étude visant à explorer les bactéries dans les ixodidae prélevés sur des animaux domestiques en Éthiopie a révélé une prévalence globale de 6% (46/767) des rickettsies de SFG, 3,8% (29/767) ADN de Borrelia et 6,4% (54/842) de C. burnetii dans différentes espèces de tiques. Africae Rickettsia a été détecté dans Amblyommma variegatum, Am. gemma, Am. cohaerens, Amblyomma spp. larves, nymphes Amblyomma, Rhipicephalus (Boophilus) decoloratus et Rh. (Bo.) Nymphes de decoloratu. La prévalence de R. africae était plus élevée dans le département centrale et orientale que dans l'ouest. R. aeschlimannii a été détecté dans Hyalomma rufipes marginatum et Hy. truncatum. Notre étude est la première à détecter R. massiliae dans Rhipicephalus Praetextatus en Ethiopie. Borrelia spp. a été détecté dans Am. cohaerens, Am. variegatum, Am. gemma, les larves Amblyomma et les nymphes Amblyomma. La nouvelle Borrelia spp. détectée dans les tiques Amblyomma clustérise entre le groupe de la fièvre récurrente et le groupe Borrelia de la maladie de Lyme alors que la Borrelia sp. détectée dans Rhipicephalus tiques clustérise avec B. theileri / B. lonestari. Coxiella burnetii a été détecté dans Am. gemma, Rh. pulchellus et dans d'autres espèces de tiques, principalement en zone Borana en Oromia. Les 18 génotypes de tiques des départements du sud-est et les 20 génotypes de tiques dans les départements centraux ont été déterminés par le typage par séquençage multilocus (MST) de C. burneti. Après la création d'une base de données de références fiables des spectres obtenus à partir des jambes sur un total de 41 spécimens de tiques suivie par un test à l'aveugle avec un total de 44 spécimens de tiques, nous avons identifié 11 espèces de tiques ixodidae par la méthode MALDI-TOF-MS. Les méthodes d'identification morphologiques des tiques et MALDI-TOF ont 8 été confirmées par séquençage du gène de l'ARNr 12S d’où une identification moléculaire des différentes espèces de tiques. L'étude pour étudier les bactéries dans 303 puces prélevés sur des chiens et des chats domestiques en Ethiopie qui ont été identifiés comme étant morphologiquement Ctenocephalides felis felis, Ctenocephalides canis, Pulex irritans et Echidnophaga gallinacé montré Rickettsia felis dans 21% des puces, principalement dans Ctenocephalides felis, avec une semblable prévalence dans les puces de chiens et de chats. Notre étude est la première à signaler Bartonella henselae en C. felis de l'Ethiopie. Notre étude sur les bactéries des poux et des moutons ked (Melophagus ovinus) a révélé la présence d'Acinetobacter spp. dans M. ovinus, Heterodoxus spiniger, Bovicola ovis et Linognathus vituli. La séquence du gène rpoB partiel a révélé la présence de A. soli, A. lowffii, A. Pitti et 3 nouveaux Acinetobacter spp. dans les poux et Keds. L'identification moléculaire des poux à l'aide d'une analyse du gène 18S a confirmé les méthodes morphologiques d'identification des poux. Bartonella melophagi a été identifié par une PCR standard, suivi par un séquençage du fragment de la gltA et gène rpoB chez M.
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