Chair of Comparative Tropical Medicine and Parasitology Faculty of Veterinary Medicine of the Ludwig Maximilians-University Munich Chairman: Univ.-Prof. Dr. Kurt Pfister EPIDEMIOLOGY OF LEISHMANIOSIS IN SOUTHERN GERMANY WITH EMPHASIS ON THE FAMILY OF PSYCHODIDAE, PRIMARILY PHLEBOTOMINAE INAUGURAL-DISSERTATION for the attainment of the title of Doctor in Veterinary Biology (Dr. rer. biol. vet.) from the Faculty of Veterinary Medicine of the Ludwig-Maximilians-University Munich by Bernadett Beran from Cottbus, Germany Munich 2010 Aus dem Lehrstuhl für Vergleichende Tropenmedizin und Parasitologie der Tierärztlichen Fakultät der Ludwig Maximilians-Universität München Vorstand: Univ.-Prof. Dr. Kurt Pfister EPIDEMIOLOGIE DER LEISHMANIOSE IN SÜD-DEUTSCHLAND MIT FOKUS AUF DIE FAMILIE DER PSYCHODIDAE, PRIMÄR PHLEBOTOMINAE INAUGURAL-DISSERTATION zur Erlangung der veterinärbiologischen Doktorwürde (Dr. rer. biol. vet.) der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München von Bernadett Beran aus Cottbus, Deutschland München 2010 Gedruckt mit Genehmigung der Tierärztlichen Fakultät der Ludgwig-Maximilians- Universität München Dekan: Univ.-Prof. Dr. Braun Berichterstatter: Univ.-Prof. Dr. Pfister Korreferent/en: Univ.-Prof. Dr. Straubinger Tag der Promotion: 24. Juli 2010 Die vorliegende Arbeit wurde nach § 6 Abs. 2 der Promotionsordnung für die Tierärztliche Fakultät der Ludwig-Maximilians-Universität München als kumulative Dissertation gestaltet. Meiner Mutter Ursula Beran (1955-2001) TABLE OF CONTENTS TABLE OF CONTENTS 1. Introduction ......................................................................................................... 1 2. Literature Review ................................................................................................ 3 2.1. Family of Psychodidae ....................................................................................................... 3 2.1.1. Taxonomy ...................................................................................................................... 3 2.1.2. Morphology ................................................................................................................... 3 2.1.3 Biology ........................................................................................................................... 5 2.2. Subfamily of Phlebotominae ............................................................................................. 5 2.2.1. Taxonomy ...................................................................................................................... 5 2.2.2. Morphology ................................................................................................................... 6 2.2.3. Distribution and Habitat ................................................................................................ 8 2.2.4. Activity and Dispersal ................................................................................................... 9 2.2.5. Life Cycle ...................................................................................................................... 9 2.2.6 Feeding Behaviour ....................................................................................................... 11 2.2.7. Vectorial Competence ................................................................................................. 11 2.2.8. Phlebotomine sand flies in parts of Europe ................................................................. 12 2.3. Leishmania ........................................................................................................................ 15 2.3.1. Systematic and Morphology ........................................................................................ 15 2.3.2. Geographical Distribution ........................................................................................... 16 2.3.3. Situation in Europe ...................................................................................................... 17 2.3.4. Life Cycle .................................................................................................................... 19 2.3.5. Vectors, Transmission and Reservoirs ........................................................................ 20 2.3.6. Manifestations of Disease ........................................................................................... 21 2.3.7. Diagnostic of Leishmania parasites ............................................................................. 22 3. Materials and Methods ..................................................................................... 23 3.1. Collection of Psychodidae and Phlebotominae .............................................................. 23 3.1.1. Entomological surveys ................................................................................................ 23 3.1.1.1. Traps ..................................................................................................................... 23 3.1.1.2. Latin Square trial .................................................................................................. 25 - i - TABLE OF CONTENTS 3.1.2. Study area in Germany ................................................................................................ 26 3.1.3. Study area in Italy ....................................................................................................... 29 3.2. Data of Climate ................................................................................................................. 32 3.3. Laboratory Examination ................................................................................................. 33 3.3.1. Identification of Psychodidae ...................................................................................... 33 3.3.2. Identification of Phlebotominae .................................................................................. 33 3.4. PCR for detection of Leishmania spp. ............................................................................. 34 3.4.1. DNA-Extraction .......................................................................................................... 34 3.4.2. Positive control and standard curve ............................................................................ 34 3.5. Retrospective study .......................................................................................................... 36 3.6. Statistical Analysis ............................................................................................................ 36 3.7. Image Acquisition ............................................................................................................. 37 4. Results ................................................................................................................. 38 4.1. Publication ........................................................................................................................ 38 4.2. Further results of the collection of Psychodidae and Phlebotominae ......................... 48 4.2.1. Latin Square ................................................................................................................ 48 4.2.2. Field collection in Southern Germany ........................................................................ 52 4.2.3. Field collection in Italy ............................................................................................... 54 4.3. Data of Climate ................................................................................................................. 56 4.4. PCR .................................................................................................................................... 56 4.5. Retrospective study .......................................................................................................... 58 5. Discussion ........................................................................................................... 62 5.1. Entomological Survey ...................................................................................................... 62 5.1.1. Psychodidae ................................................................................................................. 62 5.1.2. Phlebotominae ............................................................................................................. 63 5.2. PCR .................................................................................................................................... 69 5.3. Retrospective data acquisition ........................................................................................ 70 - ii - TABLE OF CONTENTS 6. Summary ............................................................................................................ 73 7. Zusammenfassung ............................................................................................. 74 8. References .......................................................................................................... 76 9. Abbreviations ..................................................................................................... 97 10. Figures .............................................................................................................. 99 11. Tables .............................................................................................................. 101 12. Annex .............................................................................................................