| Abstract |
The leishmaniases are a group of infectious diseases affecting humans, wild and domestic animals, and are distributed throughout all tropical and subtropical continents, except Australia. These diseases are caused by parasites of the genus Leishmania (Protozoa, Trypanosomatidae) and are transmitted by Phlebotomine sandflies (Diptera: Psychodidae). With 21 species of human-infective parasites, numerous reservoirs and vector species in a wide range of topographically different foci, the ecology and epidemiology of leishmaniases are without doubt the most diverse of all vector-borne diseases. Epidemiological studies of leishmaniasis begin with efforts to identify the vector. This is a difficult task that requires specialised training, since species are differentiated by minute morphological and anatomical traits, with character states often overlapping in related species. For this reason, and taking into account the limitations posed by all available methods (morphometrics, isoenzyme electrophoresis and radioactive DNA probes), the necessity of developing a new technique for typing of sandflies was manifested. In the first part of this work, a simple and reliable technique was developed to distinguish phlebotomine sandflies by restriction fragment length polymorphism of PCR-amplified (PCR-RFLP) small subunit (SSU) nuclear ribosomal DNA (rDNA). Specific patterns were obtained by double restriction digestion of amplified SSU rDNAs for all 10 sandfly species collected in Greece and Cyprus during epidemiological studies. Distinct RFLP profiles were also obtained for other three species that originated from Spain, India and Mexico. Furthermore, this methodology was successfully applied on sandflies for which morphological characters were badly distinguished due to minor differences between close species or to poor storage conditions, as well as on larval stage samples. This technique will allow the identification of sandfly species without the need of experts on insect morphological characters. During epidemiological studies of vector-borne diseases, it is very important to consider jointly the phylogenetic relationships among the different species, subspecies and strains of the pathogen, the host and the vector, and their interactions. Studying the impact of the genetic variability on the transmission and pathogenicity of leishmaniasis, it will be possible to explore in depth the coevolution phenomena of the links involved in the epidemiological transmission chain. For this purpose, the study progressed beyond PCR-RFLP sandfly identification to analyse phylogenetic relationships between the different species. Phylogenetic analysis explores the past of the species under study on an evolutionary scale, and can inform of possible isolation of sandfly populations, which is a critical aspect for the epidemiology of leishmaniasis. Relationships among 70 phlebotomine specimens (which belonged to 15 species and 3 genera) collected during epidemiological studies in Greece and Cyprus, were inferred from the phylogenetic analysis of SSU nuclear rDNA sequences, obtained by cloning of amplified full-length genes. Outgroups included 15 non-psychodid dipterans, and single representatives of 4 other insect orders. Within Phlebotominae, relationships were resolved at subgenus and species level. Subgenus Euphlebotomus appears to be the basal clade to Larroussius and Adlerius subgenera, which differs from previous morphological classifications. The significance of this observation becomes epidemiologically relevant since these three subgenera contain most of the Old World vectors of the Leishmania donovani complex. Data concerning epidemiology of leishmaniasis have changed during the last decades in the Old World, particularly in the Mediterranean Basin where visceral leishmaniasis was traditionally zoonotic. Leishmaniasis has become an opportunistic disease since immunocompromised patients, such as HIV patients, can serve as human reservoirs. Under these circumstances, suitable tools should be developed for the study of Leishmania epidemiology. Molecular assays for the diagnosis of leishmaniasis have been developed based on the amplification of several DNA targets, since the routine diagnostic techniques fail to detect infections in immunocompromised patients. Investigation of the possible similarities between insect and patient parasites is also very important for the epidemiological studies of leishmaniasis. The third part of the present study describes a semi-nested PCR assay for the detection of Leishmania within sandflies, which was developed based on the kinetoplastic minicircle sequence of Leishmania donovani. This methodology is more sensitive than the standard PCR since it is able to detect as few as 3 parasites per single naturally infected sandfly. Leishmania DNA was found in some samples of the species Phlebotomus neglectus (subgenus Larroussius), the only proven vector of L. infantum in Greece, and in other species, such as P. tobbi, P. simici, P. alexandri and P. papatasi. Phlebotomus tobbi, the other Larroussius species, is considered as a suspected vector of L. infantum, while the other three species (P. simici, P. alexandri and P. papatasi) have never been incriminated for the transmission of Leishmania in Greece. However, P. simici and P. alexandri are proven vectors of L. infantum and L. donovani respectively, in different regions of China. These facts, combined with the close genetic relationship shown for Larroussius and Adlerius subgenera, might suggest vectorial capacity of P. simici in Greece. The application of this technique is crucial for the identification of potential vector species and latent infections present in populations. In addition, it will permit the comparison of Leishmania DNA sequences amplified from sandfly vectors with those from patients, in order to investigate the transmission patterns of Leishmania in different endemic areas. Furthermore, the ability of these techniques to detect the presence of parasites in clinical samples, has proved useful for the diagnosis of leishmaniasis in immunocompromised patients as well as for the assessment of patients during therapy.
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Διερεύνηση της Γενετικής Ποικιλομορφίας των Αρθροποδών Διαβιβαστών της Λεϊσμανίας και Ανάπτυξη Μοριακών Επιδημιολογικών Μεθόδων
