This cross-sectional study was conducted in three active HAT foci located in the extreme southern part of Chad. These HAT foci include the Maro, Mandoul and Moissalla (figure 1).
- The HAT focus of Mandoul (8° 6'57''N; 17°06' 58''E) was previously called the Bodo HAT focus . It is located at about 50 km from Doba, the capital of “Logone Oriental” region. It has 45 villages and belongs to areas showing low risk for HAT [26-27]. Its temperature varies between 22 and 38° C and the average annual rainfall is 1000 mm . The landscape is mainly dominated by forest galleries and wooded savannah that provided favorable conditions for tsetse flies. The inhabitants of this focus practice peasant farming around the forest galleries where they build their huts or houses. The main agricultural activities are cotton, millet and sesame cultivation. Inhabitants also practice extensive animal breeding (cattle, sheep, goats, pigs and some equines). During the dry season, the Mandoul River offers a meadow to many Bororos herders in transhumance.
- The Mara HAT focus (8°28’33’’N; 18°46’10’’E) is located at 55 km from Sarh, the capital of the “Moyen Chari” region. It contains 33 villages and belongs to foci showing moderate risk for HAT . Most of these villages are located near the great Sido River. Its temperature varies between 25 to 38° C and the precipitation varies between 800 mm to 1300 mm. The vegetation is made up of savannah and clear forests with dotted trees. This vegetation offers favorable environmental conditions for the reproduction and survival of tsetse flies. Inhabitants of this HAT focus practice peasant agriculture with millet and cassava cultivation being the most predominant agricultural activities. They also practice fishing, gathering, hunting and animal breeding (cattle, sheep, goats, pigs and horses). The presence of nomadic pastoralists such as Bororo and Arabs leads to a very large cross-border movement of populations between Chad and the Central African Republic.
- The Moissala HAT focus (8°20'25''N; 17°45'58''E) is part of the great historical HAT focus of Middle Chari . It extends on the left and the right sites by Nana-Barya, along the complicated network of Moula, Dou and Bahr Kô, between Bahr Sara (Ouham) and Chari. It is located in the South of Koumra, the Capital of the Mandoul region, at about 400 km of the Central Africa Republic border. It has 25 villages and belongs to foci showing moderate risk for HAT . The temperature varies between 24 and 38° C and the average annual rainfall is about 1100 mm. The vegetation is formed by forest galleries which offer favorable conditions for the reproduction and development of tsetse flies. Inhabitants of this HAT focus practice peasant farming cotton, millet and sesame cultivation. They also practice extensive animal breeding (cattle, sheep, goats, pigs and some equines).
In the three HAT foci, the majority of inhabitants were traditional small farm holders practicing small scale animal husbandry. Sheep and goats are usually reared together with cattle. Donkeys and horses are commonly used for transportation and traction. The grazing system is essentially free grazing.
The protocol of this study was approved by the Bioethics Committee according to the decree: N° 462/PR/PM/MESRI/SG/CNBT/2017. Subsequently, the review board of the molecular parasitology and entomology unit of the Department of Biochemistry of the Faculty of Science of the University of Dschang gave its approval. Two weeks before the sampling, a sensitization mission was performed in each HAT focus. During each mission, the local administration, the religious and traditional authorities of each HAT focus were informed and the objectives of the study were explained in detail. These authorities gave their approval before all samples collection. Verbal consent was obtained from all farmers whose animals were included in the study after a detailed explanation of the study and its objectives.
Sample collection, immunologic and parasitological analyses
Donkeys and horses were sampled during field surveys in three active HAT foci of Chad. The first survey was performed from April to May 2018 and the second one in 2019 during the same period. Before each survey, the objective of the study was re-explained to inhabitants and local authorities of the villages. One day before the sampling, the inhabitants were asked to restrain and/or keep their animals. In each village, all donkeys and horses that had spent at least 3 months in the study zone were selected. From each animal, about 5 ml of blood were collected into EDTA coated tubes. The collection was performed from the jugular vein in horses and donkeys. The tubes were labelled and carefully packed to avoid crossed contamination. All horses sampled in this study were of “Poney du Logone” or “Poney Musey” breed while donkeys were local breed .
Capillary tube centrifugation test, as described by Woo , was performed on each blood sample to search for trypanosomes. To identify animals that had been in contact with T. b. gambiense, the gHAT rapid diagnostic test (RDT) was performed in parallel as described by Matovu et al. . The RDT named SD BIOLINE HAT was used in this study. It was developed using native VSGs (Nat-LiTat 1.3 and Nat-LiTat 1.5) obtained from the Institute of Tropical Medicine (ITM) in Antwerp, Belgium . It detects anti-VSG LiTat 1.3 and anti-VSG LiTat 1.5 antibodies [32, 33, 34].
At the end of immunological and parasitological tests, the remaining blood samples were centrifuged at 13,000 rpm for 5 minutes. The buffy coat was transferred into 1.5 mL micro-tubes and stored in an electric cooler. The buffy coat samples were transported, in the electric cooler, to the molecular parasitology and entomology unit of the Department of Biochemistry of the Faculty of Science of the University of Dschang in Cameroon. They were stored at -20° until DNA extraction for molecular analyses.
During sampling collection, each animal was examined by a veterinarian and its clinical status recorded.
Extraction of genomic DNA
Genomic DNA was extracted from each buffy coat sample using the Cethyl Trimethyl Ammonium Bromide (CTAB) method. Briefly, 500 µL of buffy coat and 1 mL of nuclease free water were mixed in a 2 mL micro-tube. The mixture was vigorously homogenized and then centrifuged at 11,000 rpm for 15 minutes. The supernatant was removed and 600 µL of CTAB buffer (CTAB at 5%; 1 M Tris, pH 8; 0.5 M EDTA, pH 8; 5 M NaCl) was added to the resulting pellet. This latter was re-suspended and incubated in a water bath at 60°C for 30 min. To the content of each micro-tube, 600µL of chloroform/isoamyl alcohol (24/1) mixture were added. Each micro-tube was slowly homogenized for 15 min and the upper aqueous phase was removed and transferred to a new 1.5 mL micro-tube. DNA was precipitated by adding 600 µL of isopropanol. The mixture was gently homogenized for 5 min and then incubated overnight at -20° C. After this incubation, each micro-tube was centrifuged at 13,000 rpm for 15 min. DNA pellet was then washed twice with cold 70% ethanol and dried overnight at room temperature. The resulting DNA pellet was re-suspended in 50 µL of sterile nuclease-free water and then stored at -20° C until use.
Molecular identification of different trypanosome species
Trypanosome identification was achieved by amplifying the internal transcribed spacer 1 (ITS1) of ribosomal DNA of different trypanosome species as described by Ravel et al. . For this identification, two PCR rounds were performed; the first round was carried out in a final volume of 25 µL containing 1X PCR buffer [10 mM Tris - HCl (pH 9.0), 50 mM KCl], 2 mM MgCl2, 1 µL (10 picomoles) of each primer (5'-CAAATTGCCCAATGTCG-3'/5'-GCTGCGTTCTTCAACGAA-3'), 0.5 µL (200 mM) of dNTPs, 1 µL (one unit) of Taq DNA polymerase (New England Biolab 5 U/µL), 5 μL of DNA extract and 14 µL of nuclease free water. The amplification program began with a denaturation step at 94° C for 3 minutes and 30 seconds followed by 30 amplification cycles; each of these cycles contained a denaturation step at 94° C for 30 seconds, an annealing step at 58° C for one minute, and an extension step at 72° C for one minute followed by a final extension at 72° C for 5 minutes.
The amplified products of the first PCR round were diluted 10 fold and 3 µL of each dilution was used as template for the second PCR round. This latter was performed with two other primers (5'-CCTGCAGCTGGATCAT-3'/5'-ATCGCGACACGTTGTG-3'). The amplification program was similar to that of the first PCR round. After the nested PCR, amplicons were separated by electrophoresis on 2% agarose gel that was subsequently stained with ethidium bromide and visualized under UV light.
Different trypanosome species were identified on the basis of length polymorphism of their ITS1 fragments. For instance, T. congolense strains generate DNA fragments of around 650bp (630bp for T. congolense forest and 610bp for savannah) while fragments of about 150bp and 400bp were respectively expected for T. vivax and all trypanosomes belonging to the sub genus Trypanozoon (T. brucei s.l., T. evansi, T equiperdum).
Identification of Trypanosoma congolence forest and Trypanosoma congolence savannah
Following the amplification of ITS1 sequences, all samples that showed DNA fragment between 600 to 650 bp, corresponding to the expected size of T. congolense species, were subjected to another PCR where specific primers were used to identify T. congolence forest “type" or T. congolence savannah “type". These specific identifications were done as described by Simo et al.  using TCF1 (5'-GGACACACGCCAGAAGGTACTT-3') and TCF2 (5'-GTTCTCTCGCACCAAATCCAAC-3') primers for T. congolence forest “type"  and TCS1 (5’-CGAGCGAGAACGGGCAC-3’) and TCS2 (5”-GGGACAAACAAATCCCGC-3’) primers for T. congolense savannah “type” . PCR reactions were carried out in a final volume of 25 μL containing 1x PCR buffer [10 mM Tris - HCl (pH 9.0), 50 mM KCl], 3 mM MgCl2, 1 µL (15 picomoles) of each primer, 0.5 μL (200mM) of dNTPs, 1 μL (one unit) of Taq DNA polymerase, 3 µL of DNA extracts and 16 μL of sterile water. The amplification program was comprised of a denaturation step at 94° C for 3 min 30 s, followed by 40 amplification cycles comprising, for each cycle, a denaturation step at 94° C for 30 seconds, a hybridization step at 60° C for one minute and elongation step at 72° C for 1 minute. A final elongation was done at 72° C for 5 minutes.
The amplified products were separated by electrophoresis on 2% agarose gel containing ethidium bromide (0.3 μg / ml). The DNA bands were visualized under ultraviolet (UV) light and then photographed.
Search for Trypanosoma brucei gambiense
This was done only on samples that showed a DNA fragment of about 400 bp corresponding to the expected size of trypanosomes belonging to the subgenus Trypanozoon (T. b. brucei, T. evansi, T. b. gambiense, T. b. rhodesiense). On these samples, T. b. gambiense was identified as described by Cordon-Obras et al. . This was done using a nested PCR with two pairs of primers specific to T. b. gambiense. The primer pairs TgSGP1 (5’GCT GCT GTG TTC GGA GAG C-3’ and TgSGP2- (5’-GCC ATC GTG CTT GCC GCT C-3’) described by Radwanska et al.  (2002) and, TgsGPs (5’-TCA GAC AGG GCT GTA ATA GCA AGC-3’) and TgsGPas (5’-GGG CTC CTG CCT CAA TTG CTG CA-3’) designed by Morrison et al.  were used.
The first PCR round was carried out in a total volume of 25 µl containing 2.5 µL of 10X PCR buffer [Tris - 10 mM HCl (pH 9.0), 50 mM KCl, 3 mM MgCl2], 1 µL (15 picomoles) of each of primers (TgSGP1/2), 0.5 µL (100 mM) of dNTPs, 1 µL (one unit) of Taq DNA polymerase, 5 μL of DNA extract and 14 μL of sterile water. The amplification program contained an initial denaturation at 95° C for 3 minutes followed by 45 cycles of 95° C for 30 seconds, 63° C for 1 minute and 72° C for 1 minute. A final elongation was done at 72° C for 5 minutes. Amplified products of the first PCR were diluted 10 times and 5 µL of each dilution were used as DNA template for the second PCR round. For this latter, primers TgsGPs and TgsGPas were used and only 25 amplification cycles were performed in the same conditions as for the first PCR round.
The amplified products were separated by electrophoresis on 2% agarose gel containing ethidium bromide (0.3μg/ml). DNA bands were visualized under ultraviolet (UV) light and then photographed.
Statistical analyses were performed to compare the trypanosome infection rates between animal species and HAT foci. This was done using the XLSTAT 2016 software. The Chi2 test (χ2) was used to compare, between animal species and different HAT foci, the infection rates of different trypanosomes. The threshold for significance was set at below 5%. To estimate the concordance between results generated by the tests used to identify different trypanosome infections, the kappa coefficient was determined according to Cohen , and interpreted as described by Altman .