African Trypanosomosis is a major neglected tropical disease of animals and humans in low resource settings in Africa. The disease is cyclically transmitted by Glossina (tsetse flies) spp. and mechanically by biting flies such as Tabanus spp. and Stomoxys spp. and has enormous negative effects on the health and life of both humans and animals. The socio-economic and health impact of the disease is often felt at the edge of protected, tsetse-infested wildlife areas. In Rwanda, tsetse flies and trypanosomosis are reported in areas around the Akagera National Park but the situation has previously not been well documented. This study aimed at determining (i) the distribution of species of Glossina (ii) the Trypanosoma species circulating in tsetse flies, their infection rate, and the endosymbionts, (iii) the hosts’ preference for the tsetse flies, and (iv) the Trypanosoma species circulating in cattle, at the wildlife-livestock interface of Akagera National Park in Rwanda. To determine the distribution of Glossina, a longitudinal stratified sampling entomological survey was carried out inside the park and its surroundings. Biconical traps were deployed in 55 sites for six consecutive days of each study month from May 2018 to June 2019 and emptied every 48hours. Flies caught in the traps were identified using FAO keys for entomological taxonomy. The number of flies per trap per day (FTD) was used to determine the apparent density (AD) of the flies. Pearson chi-square (χ2) and parametrical tests (t-test and ANOVA) were used to determine the variability between the variables. Logistic regression was used to determine the association between predictors of tsetse flies occurrence. A selected sample of 1101 tsetse flies, recovered from the traps, was analysed for trypanosome species and endosymbionts using PCR, and 2-gene High Resolution Melting analysis for blood meal source. A total of 1037 blood samples collected between March and July 2019 from randomly selected cattle (local and local x Friesian breeds) in four districts neighboring the National Park, and were examined for species of Trypanosoma. Four districts viz. Kayonza, Gatsibo, Nyagatare and Kirehe were selected for their proximity to the park and for being adjacent to protected game reserves in Tanzania. The presence of trypanosomes in the blood samples was determined by microscopy, immunological rapid test, and PCR coupled with High-Resolution Melt (HRM) analysis. Sanger sequencing was done on the amplicons to complement the analysis. The Cohen Kappa test was used to compare the level of agreement between the diagnostic methods. Thirty-nine thousands and five hundreds sixteen (39,516) tsetse flies were collected using the traps, of which 73.4% (29,019) and 26.6% (10,497) were from inside the park and the interface area, respectively. Female flies accounted for 61.3% (24,223) while 38.7% (15,293) were males. Two species of Glossina, i.e. G. pallidipes [n=29,121, 7.4 flies/trap/day (FTD)] and G. morsitans centralis (n=10,395; 2.6 FTD) were identified. The statistical difference was significant between the two species (p=0.000). The flies were more abundant during the wet season (15.8 FTD) than the dry season (4.2 FTD) [p=0.000]. Large numbers of flies were trapped around the swamp areas (69.1 FTD) inside the park and in Nyagatare District (11.2 FTD) at the interface [p=0.000]. One thousand and one hundred and one (1101) tsetse flies (771 Glossina pallidipes and 330 Glossina morsitans centralis) were analysed for trypanosome infections. The overall infection rate was 13.9% (153/1101) in the head and proboscis (HP) and 24.3% (268/1101) in thorax and abdomen (TA) of the flies. Eight species of trypanosomes were identified. For each species, head +proboscis and thorax+ abdomen were analyzed in parallel and are presented as HP/TA. Of these species, Trypanosoma (T.) brucei brucei accounted for 4.1/7.1%, T.congolense Kilifi (2.2/2.1%), T.congolense savannah (1.6/1.2%), T. evansi (0/0.9%), T.godefreyi (1.2/3.1%), T. grayi (0/1.08%), T.simiae (2.08/3.7%), T.theileri (0/2.08%) and T.vivax (5.2/3.7%). Mixed infections were 2.2/0.8% (25/9). No T.brucei rhodesiense was found in tsetse flies analyzed. The endosymbionts found in tsetse flies were the bacteria Sodalis (2.8%; 31/1101) and Wolbachia (4.8%; 53/1101). No Spiroplasma and SGH Virus were found in all samples analyzed. The preferred hosts for blood meal by the tsetse flies were buffalo (36.5%), warthog (14.1%), cattle (10.6%), savannah elephant (8.7%), bushbuck (7.3%), and human (5.7%). The overall prevalence of trypanosome infections in cattle was 5.6%, 7.1%, and 18.7% by thin blood smear, Buffy coat technique, and PCR/HRM, respectively. Microscopy showed a low sensitivity (28.9%) while the rapid test (VerY Diag) showed a low specificity (32.5%). Trypanosomes were detected in cattle blood, including species that are pathogenic to cattle (i.e. T. brucei brucei, T. congolense savannah, T. evansi and T. vivax) and T. theileri which is nonpathogenic. T. congolense was the most prevalent (10.7%), followed by T. vivax 5.2%, T. brucei brucei 2%, and T. evansi 0.7% by PCR/HRM analysis. Lower trypanosome infections were observed in Ankole Friesian cross-breeds than indigenous Ankole. No human-infective T. brucei rhodesiense was detected. There was no significant difference between the mean PCV of infected and non-infected animals (p>0.162). The study results on tsetse distribution, endosymbionts, hosts preference, and trypanosomes infections, corroborate other similar regional findings. Glossina pallidipes were found in higher numbers and therefore conceivably the most important vectors of trypanosomes. This study confirms that the cattle reared around the Akagera NP are infected by Trypanosoma species causing African Animal Trypanosomiasis (AAT), and the area should, therefore, be targeted in control activities. Future studies should focus on the AAT impact assessment on cattle production and information on the use of trypanocides to help policymakers prioritize target areas and optimize intervention strategies.

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