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Identifier 000435737
Title Μελέτη της εποχιακής κατανομής των ειδών Phlebotomus που απαντώνται στη Κρήτη και του ρόλου τους στη μετάδοση του πρωτόζωου παράσιτου leishmania, και μελέτη της διατήρησης της επίκτητης αντοχής του παράσιτου στα φάρμακα μέσα στους ξενιστές του
Alternative Title Study of the seasonal distribution of Phlebotomus species found in Crete and their role in the transmission of Leishmania protozoan parasite ,and study of the maintenance of acquired parasite drug resistance in its hosts
Author Τσιριγωτάκης, Νικόλαος
Thesis advisor Αντωνίου, Μαρία
Καραγωγέως, Δόμνα
Μυλωνάς, Μωυσής
Reviewer Γκίκας, Αχιλλέας
Κοφτερίδης, Διαμαντής
Πουλακάκης, Νικόλαος
Διάκου, Αναστασία
Abstract Phlebotomine sandfly is the host of protozoan Leishmania that transmits leishmaniases. It was discovered in Greece in 1910, almost a decade since the term “Leishmania” was started to be used. Distribution of Phlebotomes covers all five continents and it includes tropical and sub-tropical regions, while some species can also be found in more temperate climates. The continuously increasing mean earth temperature is a cause of concern as more regions will have favourable conditions for sandfly reproduction and survival. Apart from mountains, climatological conditions are the only barrier that prevents their distribution in inland. In general, sandflies rest and reproduce in small cracks of rocks or walls, in caves and tree hollows, and in habitats with high vegetation and animal faeces. Out of the 31 Phlebotomus species that serve as host of Leishmania, those of major medical importance in Greece are Phlebotomus (Larroussius) tobbi, Phlebotomus (Paraphlebotomus) sergenti, Phlebotomus (Paraphlebotomus) papatasi, Phlebotomus (Larroussius) perniciosus, Phlebotomus (Larroussius) neglectus, as well as the suspect host Phlebotomus (Paraphlebotomus) similis. In Crete, Phlebotomus neglectus is vector of Leihsmania infantum that causes canine leishmaniasis and visceral leishmaniasis in humans. Phlebotomus similis is a suspected vector of Leishmania tropica, that causes anthroponotic cutaneous leishmaniasis, because it is a sister species with Phlebotomus sergenti, a verified vector of Leishmania tropica. Given the increasing leishmania cases in Greeece and especially in Crete, it is important to build the knowledge on the ecology of phlebotomes that transmit leishmaniases. Leishmaniasis is a disease caused by the protozoan – parasite of genus Leishmania and infects both mammals and reptiles. Phlebotomine sandflies are the main hosts that transmit leishmaniasis. There are three types of leishmaniasis: visceral, cutaneous and mucocutaneous. Every year 0.7 – 1 million new cases are recorded in 98 countries, while mortality occurs in 3.3% of cases. In Greece, symptoms of the illness are known even before the name “leishmaniasis” was given. An increase of the cases in Greece, especially in Crete has been observed over the last few years. The most important feature of Leishmania is the acquired multidrug resistance that develops, which is induced by the resistance that develops from a single drug (MDR phenotype). Leishmania presents exceptional plasticity in genome level and is mostly characterized by aneuploidy. The population of the parasite has the ability to transform in a very short time either in gene level, or in chromosomal level. Although new drugs are added in the medical arsenal against leishmaniases, it is very important to know if acquired MDR phenotype is inherited as the parasite transforms between amastigote and promastigote form. The aim of this dissertation was: 1. the study of seasonal distribution of Phlebotomus species that exist in Crete in order to collect important data for the transmission of protozoan – parasite Leishmania. 2. investigating if the acquired resistance, using Glucantime, is inherited or not, as the parasite continues its life cycle through different hosts (mammal – phlebotomine sandfly). Fodele, a village near Heraklion in Crete, where leishmaniasis has been reported in the past, was chosen for this research. Trapping of phlebotomes was held using CDC Miniature Light Traps (John W. Hock Co., Gainesville, FL, USA) and Sticky Traps for three days in the middle of every month for three continuous years. Sampling period covered the whole period that phlebotomes remain active. The first year, sampling was not possible to begin before June. The collection of environmental data was held by ten dataloggers that measured temperature, relative humidity and dew point, as well as an anemometer. Other environmental factors, like precipitations and moon phase, were recorded too. For the research of resistance, five “resistant” and five “susceptible” Leishmania infantum isolates were chosen, that were characterized in our laboratory using Flow Cytometry. The rate of efflux of the drug in 120 minutes gives the magnitude of resistance of Leishmania (slope α). All ten isolates where measured again after they were defrost and they injected in mice. Fifteen days later, mice were euthanized and their liver and spleen were cultured, in order to recover parasites. These parasites were measured again using flow cytometry. A single isolate out of these ten, was chosen to become “resistant” and to be used for the second part of the experiment. The induction of resistance was applied, using Glucantime®, in promastigotes that were kept in culture medium and in amastigotes that were grown and multiplied in the human monocytic cell line, THP-1 and in Rag1- deficient mice. No phlebotomine sandflies were used in this stage because it was already done by another research group. Induction of resistance in promastigote was not succeeded eventually. From almost 6,000 phlebotomes (60% males, 40% females) six species were captured: Phlebotomus neglectus (57.8%), Phlebotomus similis (30.4%), Phlebotomus papatasi (2.5%), Phlebotomus simici (0.1%), Sergentomyia minuta (8.8%) and the novel species Phlebotomus (Adlerius) creticus n. sp. (0,4%). Phlebotomus neglectus and Phlebotomus similis were the most dominant species. Relatice abundance of Phlebotomus similis was higher than Phlebotomus neglectus only in the first sampling year. These two species, as well as Sergentomyia minuta, were active the whole sampling period. Seasonal dynamics of Phlebotomus neglectus is characterized by two distinct peaks (May and August / Τ = 20,3οC – 27ο C, RH = 57,5% – 63,1%), while Phlebotomus similis shows two confluent peaks (June and July / Τ = 24,3οC – 27,2ο C, RH = 53,9% – 59,1%). Male and female individuals show similar trend. The peaks of both species together, clearly indicate that the highest potential risk for Leishmania infantum transmission occurs during the summer months. Sergentomyia minuta shows similar temperature and relative humidity preferences, but different seasonal dynamics pattern. Temperature &ge; 17 – 18οC signals the beginning of Phlebotomus neglectus activation period, while temperature &ge; 20 – 22οC signals the beginning of Phlebotomus similis and Sergentomyia minuta activation period. The abundance of phlebotomes was related with temperature increase (r = 0.776, p < 0.05) and relative humidity decrease (r = 0.644, p < 0.05). Both dominant species were present in all habitats and where captured in both CDC and ST traps. However, the majority was captured in CDC and mostly those that were placed in the two habitats where domesticated animals were present (hencoops and rabbits). Intense vegetation, animal faeces and rotten fruits and vegetables were present in those habitats. The majority of Phlebotomus papatasi was collected from one of those two habitats, as well as from ST traps. Sergentomyia minuta was captured mostly from ST traps (78%). 29.9% of female Phlebotomus papatasi, 23.2% of female Phlebotomus similis, 17.1% of female Phlebotomus neglectus and 15.6% of female Sergentomyia minuta were captured in different stages of bloodmeal digestion, as well as with eggs. 50% of parasitized individuals belong to Phlebotomus similis and 41% belong to Phlebotomus neglectus, while the most intense parasitism was observed to Phlebotomus papatasi (more than 20 scars in one individual). A female Phlebotomus neglectus was parasitized by a nematode. Regarding the resistance experiment, 70% of isolates showed increased resistance after defrosting, compared to that prior to frosting. 20% of isolates showed decreased resistance, whereas 10% showed the same resistance before and after they were frosted. All the “susceptible” isolates showed increased resistance. The same isolates infected Rag1- mice. Almost 100% of splene cultures showed parasites in three to fourteen days (average six). When those isolates were measured again, 70% showed reduced resistance in contrast with those before the infection, while 30% showed increased resistance. All “resistant” isolates showed decreased levels of resistance. The next step was the induction of resistance of one Leishmania isolate, using Glucantime®. The isolate that was chosen, was characterized as “susceptible” when it was cultured from the host. Following the procedure that was described above, this isolate became “resistant”. All the parasites (treated with or without Glucantime® treatment) that had infected THP-1 cells, showed reduced resistance in contrast with the primary isolate. Verapamil blocked the pumps of those that were treated with Glucantime®, so they showed reduced slope (slope β). Parasites that were not treated with Glucantime® (THP-1_C) influxed lower amount of Rhod-123, in contrast with the primary isolate in all cases (p < 0.05). Parasites that had infected Rag1- mice, were more “susceptible” than the primary without Glucantime® treatment (Rag1_NGAI), but more “resistant” than the primary with Glucantime® treatment (Rag1_GAI). FVI differed statistically between those that were treated and those that were not treated with Glucantime® (p < 0.05).Distribution of phlebotomine sandflies is going to expand in Europe the next fifty years, towards all directions. The activation period of the adult individuals is going to increase as well. The seasonal and geographical overlap of Phlebotomus species that transmit leishmaniases in Crete, should taking into consideration by public health services, since leishmania cases are increasing. Their activation period is one of the longest in Mediterranean. Phlebotomus papatasi, even if it does not transmit leishmaniasis because of the absence of Leishmania major natural host, it has medical importance because it transmits phleboviruses. The village of Fodele is an example of rural area, like many others in Crete that offers many habitats for phlebotomine resting and reproduction. Prevention measures should include insecticides or repellents, protection dogs, given they are the natural reservoir of the parasite and appropriate information to local communities about phlebotomes and the disease. Prevention action should take place before population outbreak at the spots that phlebotomes prefer for resting and reproducing. Dogs should be vaccinated and serologically tested both in the beginning and at the end of the activation period of phlebotomes. The use of drugs against leishmaniasis should take place in an effective way. This means that if treatment is started, it should be completed. Leishmania reveals a great genetic plasticity. Aneuploidy is very common in natural populations. Isolates in laboratories, undergo such mutations that can even alter their karyotype. Freeze – defreeze cycle, as well as the completion of their life cycle through mammal host, can alter their drug resistance. Every substance that is used against leishmaniasis, causes genetic change that allows gene – targets to be found. However, these gene – targets vary between Leishmania populations, as well as between different drugs. This plasticity of Leishmania genome, reveals the reason that most drugs aim the protein pump system that is responsible for their influx and efflux of the parasite, because this system is universal. Two factors should be considered in resistance experiments. The efflux rate (slope α) that is driven by efflux protein pumps and the amount that the parasite influxes the zero time (FVI), that is driven by influx protein pumps. Their combined action will determine how much drug will remain in the parasite and for how long, which is very important, since the drug is cytotoxic. The best model organism to be used is the mammal host, because it reflects the life cycle of the parasite in nature. The results of the present dissertation show that Leishmania reacted differently in mice and this has significant implications in administering the drugs effectively. The results of this dissertation suggest that temperature plays the primary role for the beginning of the activation period of phlebotomes and is species specific, while the relative humidity is more related to their abundance. It was found that there is a specific pattern with distinct population outbreaks for each of the species of medical interest, while a novel species was discovered that is related to Phlebotomus balcanicus, which is a known transmitter of Leishmania infantum. It was also made clear that domestic animals (rabbits, poultry) should be placed at a greater distance from people's homes. On the other hand, it appeared that the parasites that show greater resistance, influx more drug than the sensitive ones. Both the freeze – defreeze cycle and the passage through the host (THP-1 cells, Rag1- mice) can affect the resistance of the parasite. The best model organism in resistance experiments is the mammal. Inside mammals, the parasites gained resistance in contrast with the primary isolate, answering the thesis question in the affirmative: the acquired resistance in the mammalian host will be maintained when Leishmania continues its life cycle.
Language Greek
Subject Glucantime
Leishmania infantum
Phlebotomus neglectus
Phlebotomus papatasi
Phlebotomus similis
Rag 1
Slope A
Slope B
Εποχιακή διακύμανση
Κυτταρομετρία ροής
Issue date 2021-03-29
Collection   School/Department
  Type of Work--Doctoral theses
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