Study area
This study was conducted in the districts of Amuru (02° 48' 49"N, 31° 56' 19"E) and Nwoya (02° 38'15"N, 32°00' 53"E) in northern Uganda, that borders the Murchison Falls National Park (Fig. 1). The two districts lie in the animal trypanosomiasis endemic areas and also fall within a Gambiense focus with cases of Gambian sleeping sickness reported annually [15]. Subsistence agriculture is the main economic activity of the districts, employing up to 98 % of the population. The arable land constitutes 90 % of the total land area in the districts, and is very fertile (Amuru District Development plan 2011–2016). However, during the last twenty years of prolonged insurgency, there was no form of vector control in place and less than 1 % of the land was utilized for agriculture, and as a result the area became re-invaded by tsetse and other biting flies.
The area has two rainfall periods, July to November and March to May, with a short dry spell in June and a fairly long period of dryness from December to February. The mean annual rainfall is about 1000 to 2000 mm. The average daily minimum and maximum temperatures are 25 °C and 39 °C, respectively [16]. The climate is favourable for agriculture and is conducive for the proliferation of tsetse flies, the vectors for African trypanosomiasis, with Glossina fuscipes fuscipes being a major species (99 %) (Angwech et al. unpublished observation). Both districts lie within the savannah grassland and are crossed by several rivers and streams. The population density of cattle in the area ranges between 10 and 20 individuals per square kilometer [17].
All parishes in Nwoya district included in the study border Murchison falls national park (i.e they are found in the wildlife-livestock interface), where wild animals used to wander freely in the course of the insurgency when the inhabitants lived in protected IDP camps in trading centers. Cattle are now being reintroduced as part of the national restocking and peace recovery programme of the government of Uganda. The density of livestock, as compared to game animals, is generally low. Therefore, tsetse flies largely depend on game animals for blood meals but also feed on livestock (Angwech et al., unpublished observation). Amuru’s parishes on the other hand are located in an area where the density of game animals is low and therefore, livestock constitute the main source of tsetse blood meal.
Sample size determination
The expected sample size for the number of cattle was determined using the formula provided by Kish [18] assuming a confidence interval of 95 %, a level of expected prevalence of 50 % and an assumed precision of 5 % given that there was no prior information about the prevalence of trypanosomiasis infection in the area. This approach generated an approximate sample size of 816.
Study design and sampling
A cross-sectional survey design was used to determine the prevalence and intensity of trypanosome species in cattle from September, 2011 to January, 2012. Two sub-counties of Atiak and Purongo in Amuru and Nwoya, respectively, were randomly selected for the study based on the information provided by the district veterinary departments about tsetse infestation in the area and trypanosome-attributable deaths of cattle. From these two sub-counties, nine parishes (Patira, Pawatomero, Latoro, Paromo, Pabit, Kal, Palukere, Pawel and Pacilo) were randomly selected for the study and bleeding centers were established in every parish. Cattle were categorized in to the following age groups: < 12 months; 12 to 48 months and > 48 months. Calves included in this study were only those herded together with other herds under communal grazing system. The ages of cattle were determined based on dentition [19] and information provided by the farmers. All cattle presented for bleeding at the centers were included in the study in order to realize the sample size. In total 816 blood samples (460 males, 356 females) were collected from 19 herds of cattle distributed in the nine parishes of Nwoya and Amuru districts (Patira = 81; Pawatomero = 86; Latoro = 87; Paromo = 103; Pabit = 90; Kal = 87; Palukere = 70, Pawel = 127 and Pacilo = 85).
Ethical consideration
This research was approved by the Gulu University Institutional Review Board and by the Uganda National Council for Science and Technology. Farmers accepted to participate in the study by signing the consent forms that were provided to them before the animals were bled. Bleeding was done by trained field veterinarians.
Blood sample collection
The animals used in this study were mainly local Boran short-horn zebu cattle under traditional communal grazing system. For each animal, whole blood sample was drawn into heparinised vacutainer tubes from the jugular vein. Four blood spots (each 120 μl) were made on a single Whatman® Classic FTA Card (Whatman Bioscience, Cambridge, UK) according to the manufacturer’s instructions. The spots were air-dried at ambient room temperature, and the cards stored moisture-free in silica gel awaiting DNA extraction.
Parasitological tests
Screening of all blood samples for trypanosomes was carried out using haematocrit centrifugation technique (HCT) [20], complemented with direct smears. Parasitological tests for all samples were carried out in the field immediately following blood collection. To perform HCT, three capillary tubes were filled by capillary attraction up to the ¾ mark with uncoagulated blood. Each capillary tube was then sealed at one end using plasticin, centrifuged at 10,000 rpm for 5 min to obtain the buffy coat, and examined for the presence of trypanosomes under a compound microscope (Olympus, magnification × 40). For direct smears, the capillary tubes were cut with a diamond-tipped pencil, and the buffy coat was examined microscopically to detect the presence of motile trypanosomes [21]. This was done for all HCT negative samples. All parasitologically positive animals were given a single curative dose of diminazene aceturate (Berenil®, 3.5 mg kg−1 body weight). Parasitaemia scores resulting from trypanosome infections in this study were categorized for intensity using a parasitaemia score from 1 (lowest intensity) to 6 (highest intensity), adopted from ILCA [22]. Scores were assigned as follows: 1[below 10]; 2[10–20]; 3[21–30]; 4[31–40]; 5[41–50] and 6 for more than 50 parasites observed per 50 random fields of view. These values were then organized into classes to ease the analysis and interpretation of results.
DNA extraction from FTA cards
Using a Harris 3-mm micro-punch (Whatman Biosciences Ltd.), five sample discs were cut randomly from a dried blood spot and placed into a 1.5 ml Eppendorf tube to reduce the chance of missing out trypanosome DNA. To prevent contamination between samples, punches were cleaned after every sampling using a 5 % sodium hypochlorite and 70 % ethanol. The punch was then used to cut a clean filter paper before using it on the next sample. DNA extraction was done using the Invitrogen™ PureLink Genomic DNA kit for purification of genomic DNA (Invitrogen Inc.) following the manufacturer’s instructions. DNA quality and quantity were checked using nanoDrop spectrophotometry and gel electrophoresis. The purified DNA sample was used immediately for PCR analyses or stored at −20 °C until use.
PCR amplification of DNA
All parasitologically positive and negative extracted DNA samples were subjected to Internal Transcribed Spacers (ITS)1 Polymerase Chain Reaction (PCR) amplification using ITS1 CF (5’CCGGAAGTTCACCGATATTG-3’) and ITS1 BR (5’TTGCTGCGTTCTTCAACGAA-3’) as the forward and reverse primers, respectively. These primers amplify ITS1 region of rDNA genes which are known to vary in size within trypanosome species, and therefore differentiate trypanosome species by their ITS1 sizes [23–25]. All primers were supplied by Bioneer Corporation. The PCR amplifications were performed in a total reaction volume of 20 μL containing 10 μM of each primer, the Bioneer AccuPower® PCR premix (Bioneer Corporation), and 2 μL of each DNA template. All PCR amplifications were performed with a thermal cycler (GeneAmp 9700 PCR system, Applied Biosystems). PCR cycling for ITS1 CF and BR primers involved an initial denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C each for 40 s, 58 °C for 40 s, followed by 72 °C for 90 s, and final extension at 72 °C for 5 min [25]. To ensure that results were not biased by false positives during repeated PCRs, negative controls in which DNA templates were replaced with sterile water as well as positive control DNAs (of each trypanosome species) were included in all PCR reactions. A 4.0 μL of each amplified PCR product was electrophoresed on a 1.5 % agarose gel in 0.5X TBE. A 1 Kb plus DNA size ladder (Fermentas) was included on each gel, stained in Gel Red dye, run constantly at 100 V for 45 min and visualized under ultraviolet trans-illumination. To confirm the results, the selected PCR products for positive samples were column purified using the GeneJet™ PCR purification kit (Fermentas Life Sciences) following the manufacturer’s instructions. Sequencing was done at the BecA-ILRI Hub Sequencing, Genotyping, OligoSynthesis and Proteomics (SEGoLIP) department using both the forward and reverse amplification primers. Sequences were edited in CLC Main Workbench 6.6.5 software (CLC Bio) and blasted to determine their identity. This was done particularly because there were PCR products that produced bands which did not correspond to any known pathogenic trypanosome species band size.
Statistical analysis
A Pearson chi-squared test and logistic regression analyses were used to determine the association between location, age, sex, and prevalence of trypanosome infections. The threshold for significance was 5 %. All statistical tests were performed using SPSS software (version 16.0.1, SPSS Inc., Chicago, IL, USA).
