Materials and methods
Description of the study area
Limmu Kosa is one of the districts located in the Oromia Regional State of Ethiopia Jimma Zone. It is bordered on the South by Kersa, on the Southwest by Mana, on the West by Gomma, on the Northwest by the Didessa River which separates it from the Illubabor Zone, on the north by Limmu Sekka, on the Northeast by the Gibe River which separates it from the West Shewa Zone and the Southern Nations, Nationalities and Peoples Region, on the East by Sokoru, and on the Southeast by Tiro Afeta. The District was located in the altitude ranging between 1200 and 3020 meters above sea level. The rivers located in the district include the Didesa main river basin, Awetu and the Dembi which are tributaries of the main river basin [3, 4] (Fig. 1).
Study design and methodologies
Entomological study
A total of 70 monoconical standard traps were deployed in the main Didessa River and most tributaries located in four different peasant associations namely Adis limat, Burqa gudina, Gale jimate and Busase with octenol (1-oct-3-nel), acetone and 3 weeks old cow urine baits [5]. All odors were placed on the ground about 30 cm upwind of the trap. The poles of traps were greased to prevent fly predators, mainly ants. Traps were allowed to stay at the site of deployment for a period of 48 h before collection. After 48 h of deployment, the catchments of each trap were sorted by fly species and then counted, identified and analyzed. The apparent density of the tsetse flies was calculated as the number of tsetse fly catch/trap/day (FTD) [6]. Sex of all collected flies was identified by observing the posterior end of the ventral aspect of the abdomen by hand lens and stereomicroscope hence male flies were identified by enlarged hypopygium in the posterior ventral part of the abdomen which is absent in female flies.
Fly dissection
The dissection procedure was carried out as described by the FAO Training manual for tsetse control personnel who began by removing wings and legs after wing fry analysis was performed the age of collected male flies was recorded and ovary analysis was used to determine the age of female flies similarly. Then, freshly killed tsetse flies were dissected under a dissecting microscope by using 0.9% normal saline. Trypanosome infections in dissected body parts of tsetse flies (i.e. midgut, salivary gland and mouthpart or proboscis) were observed using a compound microscope at a magnification of ×40 times [7]. Parasites detected in the midgut, salivary glands, and mouthparts (proboscis) were considered as Trypanozoon (T. brucei), those detected in the mouthparts (proboscis) and midguts were Nanomonas (T. congolense), and those found in the mouthparts (proboscis) only was considered in the group of Duttonella (T. vivax), immature infections considered when only found in the midgut. Finally, Giemsa stained smears (slides) were examined under oil immersion compound microscope (100 times magnification) for trypanosome species identification based on their morphological appearances [8, 13, 14]. The Infection rate of the parasite (IR) was calculated using the following formula [7]:
$$\text{Infection rate }\left( {\text{IR}} \right) = \frac{{{\text{Number of tsetse flies infected }} \times 100}}{\text{Total number of dissected flies}}.$$
Data analysis
Data collected from each deployed trap were coded into appropriate variables and entered in Microsoft excel, 2010 spreadsheet. All statically analyses were performed using STATA- 12 software. The Infection Rate (IR) was calculated for all data as the number of infected individuals divided by the number of individuals sampled times 100. Categorical data were analyzed by using the Chi square (c2) test of independence. In all cases, 95% confidence intervals were used and a p-value less than 0.05 were considered significant [9].