IntroductionThe recent outbreak of Human African Trypanosomiasis (HAT) in the Deme River Valley, Ethiopia, has highlighted the need for more comprehensive data on the infection rate of trypanosomes and the feeding patterns of tsetse. The study aimed to assess the infection rate of trypanosomes and the feeding patterns of tsetse flies collected using various attractants.Methodology/principal findingsTraps with various bait odors were placed randomly and rotated monthly. Anesthetized and recently deceased tsetse flies were examined to determine the infection rate using microscopy, and the origins of the blood meal were determined using the polymerase chain reaction technique. We used the Poisson regression model to analyze count data and the binary logistic regression model to assess binary outcomes (positive or negative) and infection rates. Of the 1208 tsetse flies captured, 751 (62.2%) wereGlossina pallidipes, while 457 (37.8%) wereGlossina fuscipes fuscipes. The trypanosome infection rate in tsetse flies was 17.1% (95% CI: 14.2-20.3). Of the 103 positive tsetse flies, 66% were infected withT. congolense, 24.3% withT. vivax, 1.9% withT. brucei, and 7.8% with eitherT. congolenseorT. brucei(the immature stages identified in the mid-gut). The infection rate of trypanosomes inG. pallidipes(19. 6%) was higher than that ofG. f. fuscipes(12.8%). Traps using a combination of cow urine and acetone captured tsetse flies with a higher infection rate of trypanosomes (21.2%), followed by acetone alone (18.9%), cow urine alone (12.2%), and traps without bait (6.1%). Of 107 freshly fed tsetse flies, 23.4% were fed to dogs, 8.4% to humans, 7.5% to cattle, and 4.7% to goats, including the mixed blood meals.Conclusions/main suggestionsThe high trypanosome infection rate in tsetse flies indicates an increased risk of trypanosomiasis infection. Although tsetse flies seem attracted to dogs, the potential risk of human exposure must also be considered. Further research is needed to understand the role of dogs in parasite transmission.Authors’ summaryAfrican Animal Trypanosomosis (AAT) is a significant issue in animal production in sub-Saharan African countries, including Ethiopia. The recent outbreak of HAT in the Deme River Valley, Ethiopia, has emphasized the need for more comprehensive data on the infection rate of trypanosomes and the feeding patterns of tsetse flies to understand human exposure. The current study revealed a high infection rate of trypanosomes in tsetse flies, withT. congolensehaving the highest prevalence, followed byT. vivax. Public concern is related to theT. bruceicomplex, as it is possibly the strain that causes HAT. Combining cow urine with acetone could significantly improve tsetse trapping and be used in tsetse suppression programs to reduce the risks of infectious tsetse flies. Analysis of the blood meal origins revealed that tsetse flies feed on various vertebrate animals, including dogs and humans. Urgent research is needed to understand the role of dogs in parasite transmission. It is recommended that large-scale tsetse control be implemented using baits such as fermented cow urine in combination with acetone for tsetse suppression.