MONITORING OF LEISHMANIA TARENTOLAE AND LEISHMANIA INFANTUM IN SAND FLIES AND SYNANTHROPIC REPTILES OF NORTHERN ITALY: EMERGING EPIDEMIOLOGICAL SCENARIOS?

Vector-borne diseases represent one-sixth of all infectious diseases and cause over one million deaths annually. Insect vectors can transmit pathogenic viruses, bacteria, and parasites. Among parasitic infections, leishmaniases, caused by protozoan parasites of the genus Leishmania, rank second only to malaria in terms of mortality. These parasites are transmitted to vertebrate hosts through the bites of infected sand flies (Diptera: Psychodidae: Phlebotominae). In Italy, leishmaniasis is primarily caused by Leishmania infantum, which is responsible for both the cutaneous and visceral forms in humans, as well as for the canine form. Since the 1990s, leishmaniasis, caused by L. infantum, became endemic in northern Italy. This has been associated with the movement of infected dogs and the expansion of sand fly populations due to climate change. The main vectors of L. infantum in Italy are Phlebotomus perniciosus and Phlebotomus perfiliewi. Another Leishmania species found in Italy is Leishmania tarentolae, which primarily infects reptiles and is transmitted by the sand fly Sergentomyia minuta. L. tarentolae is considered non-pathogenic to mammals and exposure to this parasite may elicit protective responses against pathogenic Leishmania infections. For this reason, screening for L. tarentolae is essential in leishmaniasis-endemic areas. However, little is known about L. tarentolae ecology and distribution in Italy, and S. minuta is often overlooked in Leishmania epidemiological studies. Despite this, S. minuta sand flies have been reported to feed on human blood and have tested positive for L. infantum DNA in molecular screenings. Synanthropic reptiles may also serve as reservoirs for Leishmania parasites that are pathogenic to humans. These reptiles are well-adapted to human environments and regularly interact with domestic animals, potentially exposing dogs and cats that prey on them to Leishmania parasites. Considering this scenario, my PhD project had two main objectives: i) To develop a highly sensitive and specific assay for the simultaneous and differential detection of DNA of the non-pathogenic L. tarentolae and the pathogenic L. infantum, in sand flies and dog blood; ii) To conduct a screening of L. tarentolae and L. infantum in sand flies and synanthropic reptiles in northern Italy, in an area where leishmaniasis is an emerging disease. The results of my PhD were presented in three scientific articles: Article 1 - Research paper [submitted to Scientific Reports] This study focused on developing a highly sensitive and specific assay for the simultaneous and differential detection of the DNA of the protozoan parasites L. tarentolae and L. infantum. Accurately detecting both species in sand flies and vertebrate hosts is crucial for understanding the epidemiology of the investigated area. Traditional diagnostic methods often suffer from limitations in sensitivity, specificity, and quantification, especially when dealing with complex biological samples that contain low concentrations of parasite DNA. To address these challenges, we used droplet digital PCR (ddPCR) to develop the assay. The method was validated using dog blood spiked with DNA from various isolates of both Leishmania species, as well as DNA extracted from wild-caught sand flies previously tested for Leishmania presence via qPCR. The method demonstrated high sensitivity, specificity, and no substantial cross-reactions. Article 2 - Research paper [submitted to PLOS Neglected Tropical Diseases] In this study, I investigated the biodiversity of sand flies and the prevalence of Leishmania spp. in sand flies and synanthropic reptiles in the Bergamo district, in an area where new cases of autochthonous canine leishmaniasis have recently been reported. The district is densely populated and includes numerous peri-urban areas where domestic, farm, and wild animals coexist. Few studies have focused on sand fly populations in the Bergamo district, and none have explored Leishmania prevalence in these vectors. I conducted a two-year survey of sand flies in peri-urban sites to better understand the area's sand fly biodiversity. Additionally, I performed a molecular screening for Leishmania spp. on female sand flies, as well as on the blood and faeces of synanthropic lizards, using conventional PCR and also the ddPCR method described in Article 1. Sand flies of the species P. perniciosus, Phlebotomus neglectus, and S. minuta were collected, and females of all three species tested positive for L. tarentolae and L. infantum DNA. Reptile samples also tested positive for both L. tarentolae and L. infantum DNA. This study was made possible thanks to the training I received from colleagues in the Parasitology and Micology laboratory of the University of Bari Aldo Moro. Article 3 - Review paper [published in Parasites & Vectors] I spent my PhD in the EntoPar laboratory, in a research group where one of the expertise is the study of immunological aspects of L. tarentolae and the biotechnological applications of L. tarentolae. I was therefore able to participate in the drafting of a review regarding L. tarentolae biotechnological applications, which presented also an overview of the biological and epidemiological aspects of the parasite. The review was published with the colleagues of the Parasitology and Micology laboratory of the University of Bari Aldo Moro. In conclusion, the results obtained during my PhD work revealed that the Bergamo district, which can be considered a model area for Lombardy and northern Italy for the study of leishmaniasis in newly and/or recently endemic zones, is characterized by the presence of sand flies and the circulation of both L. tarentolae and L. infantum in vectors and in synanthropic reptiles. The findings from my PhD research contributed to the advancement of molecular screening methods for Leishmania and provided data on the prevalence and distribution of Leishmania parasites, particularly in less-studied vectors and hosts. In addition, my research highlighted the potential emergence of a new epidemiological scenario for leishmaniasis in the context of northern Italy. Moreover, the detection method I developed will contribute to realize screenings in areas where leishmaniasis is endemic and L. tarentolae and L. infantum occur in sympatry.