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Molecular studies of circulating trypanosomes in Chad remain insufficient, particularly in a priming time of the disease elimination involving tsetse control and human screenings and treatments campaigns. Trypanosomes are responsible for Human African Trypanosomiasis (HAT) and Animal African Trypanosomiasis (AAT). Trypanosoma brucei gambiense and T. b. rhodesiense are the species responsible for the HAT forms (i.e. sleeping sickness). In contrast, T. b. brucei, T. congolense, and T. vivax are the most pathogenic African livestock species. The parasites are transmitted by bites of infected tsetse, flies of the genus Glossina, the most prominent vector for AAT and HAT. This study aimed to investigate the circulating trypanosomes, including potential zoonotic species in two HAT/AAT foci in Southern Chad, Mandoul and Maro. As for tsetse habitat, the Mandoul HAT/AAT focus represents an isolated ecological island. Tsetse control started in 2014 and human screenings and treatments campaigns were ongoing before 2014. In contrast, the Maro HAT/AAT focus is an area bordering the Central African Republic (CAR) where anti-tsetse campaigns had not started when this study was undertaken and represented an open area for tsetse reinvasions. 889 humans and 540 cattle were recruited, and 177 tsetse were caught to investigate the circulating trypanosome species in both foci. During the surveys on humans, organised in 2017 and 2018 in nomadic and sedentary settlements, questionnaires related to demographic data, anthropometric measurements, health and nutritional status, literacy, and knowledge on parasitic vector diseases were recorded, besides the blood and urine sample collections. Also, cattle blood samples were collected, and questionnaires linked to the settlement systems (nomadic or sedentary), common animal diseases, and animal health status, were filled, and the PCVs measured. During the entomological surveys, alive tsetse were dissected in wings, legs, and proboscis, and gut tissues. As an outcome, the surveyed human groups showed different responses on the social determinant of health (SDOH), on which the vulnerability to infectious diseases and non-communicable diseases depend. The low education level and the lack of sensitisation hampered the understanding of vector-borne parasitic diseases and the preventive measures to be adopted. Interestingly, only a few people are obese, or in overweight range in these areas, with few who presented a high glycemia and high blood pressure. Looking at the entomological data, tsetse are rare in Mandoul where only one fly was caught. In contrast, in Maro, their presence was farther noticed. Extracted DNA samples screened using nested PCR targeting the internal transcribed spacer I (ITS1) supported by the glycosomal glyceraldehyde- 3-phosphate dehydrogenase (gGAPDH)-PCR, and sequencing revealed different trypanosomes diversity according to focus. In Mandoul, there is no evidence of T. b. gambiense and T. congolense neither in humans nor in cattle. Since only one fly was collected here, no statement on presence of trypanosomes in tsetse can be made. However, an unknown Trypanosoma species (Trypanosoma-sp.-129-H) was identified in one person, and a high prevalence of T. theileri in cattle, besides few T. vivax and T. simiae. In contrast, in Maro, T. b. gambiense was identified in humans, as well as evidence for T. congolense DNA. Furthermore, in this focus, sample from cattle and tsetse showed evidence for a high diversity of trypanosomes. This includes pathogenic, non-pathogenic, and unknown species. The diversity of trypanosome species in Maro is linked to the relatively high number of tsetse observed. Consequently, tsetse are vectoring trypanosomes in this focus. This is influenced mainly by the missing tsetse control, besides pastoralist transhumance activities from and to CAR. Whereas in Mandoul, the campaigns are ongoing since 2014 (i.e., three years before and during the surveys). They have reduced the tsetse populations and the pathogenic trypanosome species. However, the other species identified in this focus could be transmitted by other vectors such as tabanid and Stomoxys. Regarding the evidence of T. congolense in humans, it was also confirmed when performing the ELISA assay to investigate the anti-TconTS (anti-T. congolense Trans-sialidase), an enzyme produced during infection with this species. The assay showed a high number of people having an immune response against this parasite exposure. It could be due to a current or cleared infection. The evidence of zoonotic trypanosomes in humans need to be monitored in the field, and the pathogenicity studied in case of non-transient infection. For this purpose, loop-mediated isothermal amplification (LAMP) techniques were optimised for both human and animal pathogenic Trypanosoma species. The assays were successfully tested in the field for the identification of T. congolense in cattle. This thesis gives an insight into the circulating trypanosomes in the Mandoul and the Maro HAT/AAT foci in these cited hosts. It provides a better understanding of the genetic of trypanosomes to support elimination goals. In conclusion, there is evidence of the diversity of trypanosomes and emerging zoonotic species in the study sites. Thus, we suggest widening the study to other mammalian hosts, vectors, and involving other areas, as well as joining efforts with CAR in terms of tsetse control operation and disease elimination in these areas. We also suggest investigating the potentials of emerging zoonotic trypanosomes.

Background African trypanosomiases are vector-borne diseases that affect humans and livestock in sub-Saharan Africa. Although data have been collected on tsetse fauna as well as trypanosome infections in tsetse flies and mammals in foci of sleeping sickness in Chad, the situation of tsetse fly-transmitted trypanosomes remains unknown in several tsetse-infested areas of Chad. This study was designed to fill this epidemiological knowledge gap by determining the tsetse fauna as well as the trypanosomes infecting tsetse flies in the area of Lake Iro in southeastern Chad. Methods Tsetse flies were trapped along the Salamat River using biconical traps. The proboscis and tsetse body were removed from each fly. DNA was extracted from the proboscis using proteinase K and phosphate buffer and from the tsetse body using Chelex 5%. Tsetse flies were identified by amplifying and sequencing the cytochrome c oxydase I gene of each tsetse fly. Trypanosome species were detected by amplifying and sequencing the internal transcribed spacer 1 of infecting trypanosomes. Results A total of 617 tsetse flies were trapped; the apparent density of flies per trap per day was 2. 6. Of the trapped flies, 359 were randomly selected for the molecular identification and for the detection of infecting trypanosomes. Glossina morsitans submorsitans (96.1%) was the dominant tsetse fly species followed by G. fuscipes fuscipes (3.1%) and G. tachinoides (0.8%). Four trypanosome species, including Trypanosoma vivax, T. simiae, T. godfreyi and T. congolense savannah, were detected. Both single infection (56.7%) and mixed infections of trypanosomes (4.6%) were detected in G. m. submorsitans. The single infection included T. simiae (20.5%), T. congolense savannah (16.43%), T. vivax (11.7%) and T. godfreyi (9.8%). The trypanosome infection rate was 61.4% in G. m. submorsitans, 72.7% in G. f. fuscipes and 66.6% in G. tachinoides. Trypanosome infections were more prevalent in tsetse bodies (40.6%) than in the proboscis (16.3%). Conclusion This study revealed the presence of different tsetse species and a diversity of trypanosomes pathogenic to livestock in the area of Lake Iro. The results highlight the risks and constraints that animal African trypanosomiasis pose to livestock breeding and the importance of assessing trypanosome infections in livestock in this area. Graphical Abstract