| Abstract |
Leishmaniasis is one of the most important parasitological diseases worldwide. The parasite, Leishmania, is found, among other areas, in all the Mediterranean countries and Portugal causing the visceral as well as the cutaneous form of the disease. Visceral Leishmaniasis (VL) is the most aggressive form and it is fatal, if left untreated, for the 95% of the cases. Clinical manifestations are fever, lymph and spleen enlargement. Cutaneous Leishmaniasis (CL) is mild and manifests as cutaneous ulcers.
Sand flies, genus Phlebotomus, are the vectors of the parasite. Their diet consists of plant sugars but females feed on mammal blood (such as humans) to complete egg development. During those meals, sand flies transmit parasites and other pathogens, as Bartonella sp. and Phleboviruses to humans and other animals.
In the Old World, there are 49 sand fly species of the genus Phlebotomus that act as Leishmania vectors. Different sand fly species transmit different Leishmania species. Thus, by studying the geographical distribution of sand fly species and knowing their Leishmania infection rates is possible to evaluate the risk of leishmaniasis in an area. Furthermore, the potential of disease emergence in a new area can be predicted if the sand fly species present in an area is known.
Typing Phlebotomus species is done mainly by morphology using a microscope. In addition, due to the limitations of morphological identification, molecular typing methods are developed in order to save time and to increase specificity. Nevertheless, there is no universal molecular typing method used by researchers. Phylogenetic relationships among Phlebotomus species are not well studied and, as a result, morphological typing, on its own, often offers limited information. Given that, researchers tend to have conflicting opinions regarding the taxonomy of several sand fly species. It is clear that there is a need for quick and universal molecular typing tools to be applied together with morphotyping. Using only morphological typing can lead to incorrect conclusions regarding the geographical distribution of the Phlebotomus species found in an area and hence the risks posed to human health in relation to the pathogens the particular species may transmit.
Phylogenetic relationships and species typing of Phlebotomus species from Crete and Cyprus have been remotely analyzed, therefore there is no detailed knowledge, on the species that reside and transmit Leishmania in these areas. Human and dog leishmaniasis cases in both these regions are increasing. Therefore, the knowledge on the sand fly vectors responsible for this is imperative.
This work aimed at discriminating phylogenetic relationships of sand fly species from Crete and Cyprus and to develop new, reliable molecular typing methods for doing so. Sand fly sampling was done in years 2011 - 2014 during the EU, FP7, EDENext research project. Choosing the sand flies for the analyses was done carefully in order to have the right gender ratio and sample size, for a balanced representation of the sand fly populations circulating in these islands during that period.
The DNA barcoding method was used to analyze the phylogenetic relationships of sand fly species. This process consists of sequencing a fragment of the mitochondrial gene encoding cytochrome oxidase 1 (COI). Sequences derived from that gene provided information on the molecular identity of the species studied. They also provided the ability to build, based on that information, the phylogenetic tree that defines the systematic status of each species and its relationship with others. Results, among others, revealed, for the first time, the COI barcodes for P. neglectus and P. similis, vectors of the parasites L. infantum (causing VL) and L. tropica (causing CL) in Greece, respectively.
Resolving the phylogenetic position of the sand fly species studied has provided the way to develop a molecular typing tool for Larroussius subgenus using the PCR – RFLP method. This subgenus contains vectors of the parasite L. infantum, the VL causing agent. Identification of the Larroussius subgenus members is difficult, based on current methods especially that of the females. This tool proved to be quick and reliable in typing Larroussius species from the study area.
Finally, this work helped to develop a new and promising sand fly molecular typing tool using the individual’s proteome information, i.e. the insect tissue protein composition profile. This method uses mass spectrometry (MALDI – TOF MS). The first analyses were conducted on laboratory reared sand flies and this was the first such study in the field, worldwide. This method, in the future, promises to be one of the top tools used in identification studies of sand flies and other insects.
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