1 Parasitological and molecular detection of Trypanosoma species from domestic animals in the

2 tsetse infested areas of Somalia

3

4
5 Ahmed A. Hassan-Kadle1,2*¶, Abdalla M. Ibrahim2*¶, Hamisi S. Nyingilili3, Abdulkarim A. Yusuf2, Rafael F.
6 C. Vieira1
7
8
1
9 Departamento de Medicina Veterinária, Universidade Federal do Paraná, Curitiba, PR, Brasil
2
10 Abrar Research and Training Centre, Abrar University, Mogadishu, Somalia
3
11 Vector and Vector Borne Diseases Research Institute, Tanga, Tanzania
12
13
14 *Corresponding authors:
15 Email: akadle@abrar.edu.so (AAH-K); abdallami@abrar.edu.so (AMI)
16
17
¶
18 These authors contributed equally to this work
19

20 Abstract
21 African animal trypanosomiasis (AAT) affects the health and productivity of livestock of 12.3 million Somalis
22 and severely constraints their development and wellbeing, as well as the general economy of the country. In
23 addition, a recent data on animal trypanosomiasis in the country after the civil of 1990s was missing.
24 Therefore, this cross-sectional study was conducted from November 2017 to February 2018 with a total of 614
25 blood samples collected from cattle (n = 202), sheep (n = 206) and goats (n = 206) grazing in tsetse infested
26 areas of Afgoye and Jowhar districts, Somalia for the detection and identification of Trypanosoma spp. using
27 microscopical (Buffy Coat Technique, BCT) and molecular (Polymerase Chain Reaction, PCR) techniques. A
28 total of 21/614 (3.4%; 95% CI: 2.1-5.2%) and 120/614 (19.5%; 95% CI: 16.6-22.9%) ruminants were positive
29 for AAT by BCT and nested PCR (nPCR), respectively. The highest prevalence was observed in cattle
30 (31.7%; CI: 25.3-38.6%) by nPCR followed by goats (18.4%; CI:13.4-24.4%) and sheep (8.7%; CI: 5.3-
31 13.5%) with significant variation (p=0.00000003). Animals sampled from Afgoye district were approximately
32 two times (OR=1.8; 95% CI: 1.2-2.8) more likely to be infected with trypanosomiasis than animals reared in
33 Jowhar district. The mean PCV value of trypanosome infected ruminants in BCT and nPCR results were
34 significantly low (p=0.002, p=0.001 respectively) in comparison to their test-negative counterparts. Six
35 Trypanosoma spp. were detected in this study including T. evansi, T. godfreyi, T. vivax, T. brucei, T. simiae
36 and T. congolense. A total of 94/120 (78.3%; 95% CI: 70.1-84.7%) ruminants were coinfected with at least
37 two Trypanosoma species. The T. brucei rhodesiense, human infective trypanosome, was not found in the
38 tested samples. The present study indicates that tsetse and non-tsetse transmitted animal trypanosomiasis were
39 prevalent in the studied areas. Further large-scale studies and sustainable control programmes of the disease
40 are needed.
41 Keywords: Trypanosomiasis, Glossina spp., AAT, HAT, Southwest, Hirshabelle
42

43 1. Introduction
44 African animal trypanosomiasis (AAT) is a parasitic disease caused by protozoans of the genus
45 Trypanosoma (OIE Manual 2013). The most important trypanosomes infecting animals in terms of economic
46 losses are T. congolense, T. vivax and T. brucei (Paul et al. 2018). This disease is transmitted cyclically by
1
47 several species of Glossina genus, each of which is adapted to different climatic and ecological conditions
48 (OIE Manual 2013, Ford 1970). While tsetse flies are not the only vectors of African trypanosomes but the
49 tsetse-transmitted form of the disease is the most serious problem because it remains infective for a period of
50 time once the tsetse fly itself becomes infected, in contrast to the ephemeral nature of the threat from non-
51 cyclical transmission (Murray and Gray 1984). Four species of Glossina exist in Somalia, G. austeni, G.
52 brevipalpis, G. Longipennis and the most widely distributed G. pallidipes and they are associated with forest
53 and thicket areas of Shabelle and Jubba River systems in southern regions of the country (Moggridge 1936,
54 Hursey 1985, Mohamed and Dairri 1987, Torr et al. 1989). It is estimated that a total of 9407 km2 of the
55 river valleys and 3070 km2 in southern border of Kenya is infested with one or more species of tsetse fly
56 (Harberd 1988).
57 Economic impacts of animal trypanosomiasis are inestimable and result from direct loss (mortality,
58 production losses, costs of prophylactic and curative trypanocidal drugs) and indirect losses due to losses in
59 crop production and agricultural workers (deficiency of animal protein diets) (Harberd 1988, Angara et al.
60 2014). It had been estimated to cost Africa about 4.5 billion US dollars a year (Oluwafemi et al. 2007).
61 Although little information was available on the economic impact of animal trypanosomiasis in Somalia, an
62 annual loss of approximately 88 million US dollars was estimated (Mohamed and Dairri 1987). A National
63 Tsetse and Trypanosomiasis Control Project (NTTCP), funded by the European Union, was established in the
64 1980s in Somalia (Mohamed and Dairri 1987). Apart from a tsetse and trypanosomiasis (T & T) control
65 projects in some villages of Shabelle and Jubba regions using traps and live-baits funded by the International
66 Committee of the Red Cross (ICRC) Somalia program (ICRC-blog 2017), no other control measures or wide
67 coverage area to reduce the losses from trypanosomiasis have been implemented following the collapse of the
68 central government of Somalia in 1991 (Salah 2016). Effective and sustainable T & T control projects will
69 contribute in improving livestock health which enhances the production and livelihood of dependent families
70 (Swallow, 1999).
71 Examination of thick and thin peripheral blood or buffy coat films stained with Giemsa stains or fresh
72 wet blood or buffy coat smears is used for detection and identification of species of Trypanosoma (OIE
73 Manual 2013, Rosenblatt 2009). However, these conventional parasitological techniques are often fails to
74 detect low levels of trypanosomes in the blood (Mattioli and Faye 1996, Picozzi et al. 2002) and this
75 underestimates the prevalence of trypanosomiasis in the area. Therefore, molecular methods (polymerase
76 chain reaction, PCR) overcome the low sensitivity limitations of parasitological approaches and able to
77 identify trypanosomes to the subspecies level and to detect mixed infections (Picozzi et al. 2002, Desquesnes
78 and Dávila 2002, Kouadio et al. 2014).
79 In Somalia, animal trypanosomiasis were reported in different domestic ruminants using microscopical
80 methods of diagnosis (Mohamed and Dairri 1987, Macchioni and Abdullatif 1985, Dirie et al. 1988). Dirie
81 et al., (1988) tested 40 sheep after being experimentally reared in tsetse infested area and revealed 42.5% with
82 T. congolense and 17.5% with T. vivax (Dirie et al. 1988). Moggridge (1936) have reported T. congolense in
83 cattle from Genale area of Lower Shabelle region (Moggridge 1936) and Dirie et al., (1988) recorded T. vivax
84 in cattle from Libsoma, Jilaal-Mooge and Geed-faqe areas of Lower Shabelle region (Dirie et al., 1988).
85 Human African Trypanosomiasis (HAT) or sleeping sickness has been reported in Ethiopia (Baker et
86 al. 1970) and Kenya (Rutto and Karuga 2009). Despite the presence of Glossina spp., the sleeping sickness
87 situation is unknown in Somalia (Harberd 1988, WHO 2007).
88 The widespread infestation of trypanosome vectors in Somalia is one of the main obstacles to the
89 development of the livestock industry which supports the livelihood of 12.3 million Somali communities
90 living in an area of 640,000 km2 (Bernacca 1967, UNFPA 2014). However, the data on animal
91 trypanosomiasis after the civil of 1990s was missing. Hence, the present study aimed to investigate the
92 epidemiological situation of trypanosomiasis in cattle and small ruminants in the tsetse infested areas of
93 Lower Shabelle (Afgoye district) and Middle Shabelle (Jowhar district) regions of Somalia using
94 microscopical and molecular diagnostic methods.
95

2
 96 2. Materials and methods
97 Ethics statement:
98 This study was reviewed and approved by the Ethical Committee of Abrar University, Somalia (reference
99 number AU/ARTC/EC/04/2017).
100 2.1. Study Area
101 The present study was carried out in the tsetse infested villages along the river bank of Afgoye (13° 4’
102 N and 36° E) and Jowhar (13° 4’ N and 36° E) districts, Somalia.
103 2.2. Sampling
104 A total of 614 blood samples were collected from domestic animals between November 2017 and
105 February 2018. Approximately 5 ml of whole blood was drawn from jugular vein of each animal into a
106 heparinized vaccutainer tubes. The samples were transferred to Abrar Research and Training Centre (ARTC),
107 Abrar University for sample preparation and part of laboratory investigation. Two haematocrit tubes were
108 filled three-quarters to full for the determination of packed cell volume (PCV) and parasite examination using
109 buffy-coat technique (BCT) following Murray et al., (1977) method (Murray et al. 1977). In addition, blood
110 spots on Flinders Technology Associates (FTA®) cards for molecular detection of the parasites were prepared
111 and stored in –20oC until processed.

112 2.3. Microscopical analysis

113 2.3.1. Buffy-Coat Technique (BCT)
114 Two heparinised haematocrit tubes were filled with blood from each sample and one end of each tube
115 was sealed with plasticel. The sealed capillary tubes were then centrifuged for four minutes at 12,000
116 revolutions per minute (rpm). Then, the buffy coat content was examined for the presence of trypanosomes.
117 2.3.2. Packed Cell Volume (PCV) determination
118 After centrifugation of capillary tubes, the degree of anaemia (PCV%) was recorded using haematocrit
119 reader (Shanghai surgical instruments factory, China).

120 2.4. DNA extraction and PCR processing

121 This study applied five different PCR assays to detect trypanosome species in cattle, sheep and goats:
122 (i) nested PCR (nPCR) that detects the inter-specific length variation of the internal transcribed spacer (ITS)
123 regions for each species of trypanosomes (Cox et al. 2005), (ii) TBR-PCR specific for T. brucei (Moser et al.
124 1989), (iii) SRA-PCR that targets the serum resistance associated (SRA) gene uniquely specific for T. b.
125 rhodesiense (Maina et al. 2007), (iv) RoTat 1.2 PCR that amplifies the Rode Trypanozoon antigenic type
126 (RoTat) 1.2 variable surface glycoprotein (VSG) gene fragment specific for the detection of T. evansi
127 (Urakawa et al. 2001), and (v) TSM-PCR for detection of T. simiae (Masiga et al. 1992).
128 DNA was extracted from 614 blood samples spotted on the FTA® cards using Chelex® 100 resin
129 (Sodium form, 100-200 dry mesh, Sigma–Aldrich, St. Louis, USA) procedure described by Ahmed et al.
130 (2011) and stored at –20oC until used for PCR assays.
131 Detection and identification of trypanosomes were conducted using generic and species-specific PCR
132 primers. The nested PCR (Cox et al., 2005) was carried out to identify and differentiate the infecting
133 trypanosome species basing on the size of their ITS region. Samples positive for Trypanozoon group were
134 further tested for T. brucei using TBR-PCR (Moser et al. 1989). Positive TBR samples were further screened
135 for T. b. rhodensiense by nested PCR for SRA gene (Maina et al. 2007) while negative TBR samples were
136 subjected to RoTat 1.2 PCR for the presence of T. evansi (Urakawa et al. 2001). Furthermore, all T. simiae
137 positive samples were subjected to TSM primers described by Masiga et al. (1992). The PCR primers used in
138 these molecular tests are shown in table 1. A positive control was added to the corresponding PCR method
139 and PCR grade water was used as negative control for each PCR reaction. The amplified PCR products were

3
140 analysed by electrophoresis in a 1.5% agarose gel, stained with ethidium bromide and visualized by UV
141 illuminator (UVITEC™ Cambridge, UK).
142 Table (1). The PCR primer sequences used in the present study
Taxa Specific primers Primer sequence Reference
ITS1 outer forward 5′-GATTACGTCCCTGCCATTTG-3′
Trypanosoma ITS2 outer reverse 5′-TTGTTCGCTATCGGTCTTCC-3′
Cox et al., 2005
spp. ITS3 inner forward 5′-GGAAGCAAAAGTCGTAACAAGG-3′
ITS4 inner reverse 5′-TGTTTTCTTTTCCTCCGCTG-3′
RoTat 1.2 forward (ILO7957) 5′-GCCACCACGGCGAAAGAC-3′
T. evansi Urakawa et al. 2001
RoTat 1.2 reverse (ILO8091) 5′-TAATCAGTGTGGTGTGC-3′
TSM1 forward 5′-CCGGTCAAAAACGCATT-3′
T. simiae Masiga et al. 1992
TSM2 reverse 5′-AGTCGCCCGGAGTCGAT-3′
TBR1 forward 5′-CGAATGAATATTAAACAATGCGCAGT-3′
T. brucei Moser et al. 1989
TBR2 reverse 5′-AGAACCATTTATTAGCTTTGTTGC-3′
SRA-out-s 5′-CCTGATAAAACAAGTATCGGCAGCAA-3′
T. b. SRA-out-as 5′-CGGTGACCAATTCATCTGCTGCTGTT-3′
Maina et al. 2007
rhodesiense SRA-inner-s 5′-ATAGTGACATGCGTACTCAACGC-3′
SRA-inner-as 5′-AATGTGTTCGAGTACTTCGGTCACGCT-3′

143 2.5. Data management and analysis:

144 The collected data were recorded in notebook and later entered, coded and stored in a Microsoft®
145 Excel 2016. Then, the data were analysed using Statistical Package for Social Sciences (SPSS) version 25
146 (IBM Corp., Armonk, New York, USA) and Epi Info™ Software (version 7.2.2.6). The proportions of animals
147 infected by different trypanosome species were compared between animal species and districts using either
148 the Chi-square (χ2) or Fisher’s exact test. Odds ratios (OR), 95% confidence interval and p values were
149 calculated separately for each variable. All statistical tests were considered significantly different when
150 p<0.05. Normality of the PCV value distribution was tested using the Shapiro-Wilk normality test (Ghasemi
151 and Zahediasl 2012). The data were considered not normally distributed when p<0.05.

152 3. Results
153 3.1. Microscopical detection
154 The microscopic (BCT) results indicated a total of 21/614 (3.4%; 95% CI: 2.1-5.2%) animals were
155 infected with different trypanosome species in a single and mixed infections. Most of samples had very low
156 parasitaemia and mix infection of T. brucei, T. vivax and T. congolense was observed in cattle based on their
157 movement. The highest prevalence was observed in cattle (6.9%) with a highly statistical significance
158 variation (p=0.004) compared to small ruminants (table 2).
159 Table 2: BCT prevalence of trypanosome infection in different animal hosts:
Host Number of samples BCT prevalence 95% CI P-value
Cattle 202 14(6.9%) 3.8-11.4
Sheep 206 4(1.9%) 0.5-4.9
0. 004 (χ2=11.3)
Goat 206 3(1.5%) 0.3-4.2
Total 614 21(3.4%) 2.1-5.2
160 There was statistical significance (p=0.013) in the microscopic prevalence among the districts. Similar
161 statistical significant differences were also reported within the animal species from Afgoye district (p=0.029)
162 but at the threshold significance (p=0.05) in animals from Jowhar district. Among different animal species,
163 cattle herds reported the highest prevalence in both districts (table 3).
164 Table 3: BCT prevalence of AAT in Afgoye and Jowhar districts:
State Region District Host N (%) BCT prevalence n(%) 95% CI P-value
Lower Cattle 100 10(3.3) 1.6-5.9
Southwest Afgoye 0. 029 (χ2=7.1)
Shabelle Sheep 102 4(1.3) 0.4-3.3
4
 Goat 102 2(0.7) 0.08-2.4
Sub-total 304 (49.5) 16(5.3) 3.04-8.4
Cattle 102 4(1.3) 0.4-3.3
Middle
Hirshabelle Jowhar Sheep 104 0(0) 0.0-1.2
Shabelle 0. 05 (χ2=4.3)
Goat 104 1(0.3) 0.01-1.8
Sub-total 310 (50.5) 5(1.6) 0.5-3.7
Total 614 (100) 21(3.4) 2.1-5.2 0. 013 (χ2=6.1)
165 3.2. Molecular detection
166 3.2.1. Detection of trypanosome species by nested PCR assay
167 The overall prevalence of animal trypanosomiasis by the nPCR was 19.5% (120/614; 95% CI: 16.6-
168 22.9%) with the highest prevalence observed in cattle (31.7%) followed by goats (18.4%) and sheep (8.7%)
169 (table 4). There were statistically significant differences in trypanosome infections within the districts
170 (p=0.003) and within animal species (p=0.00000003) (table 4 & 5). The risk of trypanosome infections was
171 approximately two times (OR=1.8; 95% CI: 1.2-2.8) higher in Afgoye district than Jowhar district.
172 Table 4: Trypanosome infection in different animal hosts by nPCR:
Number of nPCR
Host 95% CI P-value
samples prevalence
Cattle 202 64(31.7) 25.3-38.6
Sheep 206 18(8.7) 5.3-13.5 0.00000003
Goat 206 38(18.4) 13.4-24.4 (χ2=34.4)
Total 614 120 (19.5) 16.6-22.9
173 Table 5: AAT in Afgoye and Jowhar districts by nPCR:
State Region District Host N nPCR prevalence n(%) 95% CI P-value
Cattle 100 49(16.1) 12.1-20.7
Lower
Southwest
Shabelle
Afgoye Sheep 102 11(3.6) 1.8-6.4 0.00000000002
Goat 102 14(4.6) 2.5-7.6 (χ2=49.4)
Sub-total 304 74(24.3) 19.6-29.6
Cattle 102 15(4.8) 2.7-7.9
Middle
Hirshabelle Jowhar Sheep 104 7(2.3) 0.9-4.6
Shabelle 0.004 (χ2=11)
Goat 104 24(7.7) 5.0-11.3
Sub-total 310 46(14.8) 11.1-19.3
Total 614 120 (19.5) 16.6-22.9 0.003 (χ2=8.8)
174 3.2.2. Detection of trypanosome infection by species-specific PCR primers
175 All (91 animals) trypanozoon group were subjected to TBR and RoTat 1.2 PCR assays for detection of
176 T. brucei and T. evansi, respectively. The TBR primers detected 4/91 (4.4%) T. brucei while the RoTat 1.2
177 PCR test detected 87/91 (95.6%) animals with T. evansi infection. The TSM PCR test confirmed 89.5%
178 (17/19) of the nPCR positive samples using T. simiae specific primers.
179 3.2.3. Detection of T. b. rhodesiense in animals by SRA gene
180 The TBR positive samples were further analysed for the presence of human-infective trypanosome by
181 SRA-based PCR. No T. b. rhodesiense DNA was found in the tested animals from Somalia.
182 3.2.4. Single and mixed trypanosome infections
183 Six species of trypanosomes were identified in this study and the specific distribution of the parasites
184 is presented in Fig 1. Mixed infections were detected in 94/120 (78.3%; 95% CI: 70.1-84.7%) of the
185 ruminants. Only 26/120 (21.7%; 95% CI: 14.7-30.1%) of animals had a single infection of trypanosomes.
186 Coinfections between T. evansi and T. vivax (18/120; 15%) and triple-infection of T. evansi/T. godfreyi/T.
187 vivax (15/120; 12.5%) were the most frequent parasites recorded in the current work. These coinfections were
188 mainly observed in goats (10/120; 8.3%) and cattle (6/120; 5%) from Afgoye district, respectively. None of
189 sheep samples were shown T. simiae infection.
190
191

5
192 Fig 1. Distribution of trypanosome species in Afgoye and Jowhar districts

60

50

No. of infections
40

30

20

10

0
Afgoye District Jowhar District
T. brucei T. vivax T. godfreyi T. simiae T. congolense T. evensi
193
194 3.2.5. PCV values of infected and non-infected animals
195 The present study used 26% cut-off of PCV values as an indication for anaemia in trypanosome
196 infected animals (Marcotty et al. 2008; Abdalla et al. 2015). The PCV data differs significantly from a
197 normal distribution (W=0.99, p=0.0001) using Shapiro-Wilk test. Therefore, the Mann–Whitney test was used
198 for comparison of infected and non-infected animals. The mean PCV value of the infected animals in both
199 BCT (25.48%) and nPCR (27.02%) results were significantly low (p<0.05) in comparison to their test-
200 negative counterparts (table 8).
201 Table 8. Mean PCV of infected and non-infected animals by BCT and nested PCR techniques:
N examined Mean PCV % N examined Mean PCV %
Condition ±SD P-value ±SD P-value
by BCT (95% CI) by nPCR (95% CI)
Infected 21 25.48 (23.9-27.1) 3.49 120 27.02 (26.3-27.7) 3.78
0.002 0.001
Non-infected 593 28.21 (27.9-28.5) 4.03 494 28.38 (28.0-28.7) 4.06

202 The proportion of animals in the PCV categories (≤ and >26%) for both BCT and nPCR are delineated
203 in table (9) and the distribution of the PCV values are shown in Fig 2. Out of 21 infected animals by BCT,
204 62% (13/21) had a PCV value ≤26% with statistical significance of p=0.012, whereas 56% of the nPCR
205 positive animals had a PCV higher than 26% with statistical significant (p=0.038).
206 Table 9: Proportion of animal species at 26% cut-off PCV-value by BCT and nested PCR methods:
Tests BCT results nPCR results
Species PCV categories P-value P-value
PCV ≤26% PCV >26% PCV ≤26% PCV >26%
Test results
Positive 64% (9/14) 36% (5/14) 0.000 56% (36/64) 44% (28/64) 0.000
Cattle
Negative 51% (95/188) 49% (93/188) (χ2 = 92.5) 49% (68/138) 51% (70/138) (χ2 = 202)
Positive 75%( 3/4) 25% (1/4) 0.000 44% (8/18) 56% (10/18) 0.000
Sheep
Negative 30% (60/202) 70% (142/202) (χ2 = 37.6) 29% (55/188) 71% (133/188) (χ2 = 111)
Positive 33% (1/3) 67% (2/3) 0.000 24% (9/38) 76% (29/38) 0.000
Goat
Negative 26% (53/203) 74% (150/203) (χ2 = 31.2) 27% (45/168) 73% (123/168) (χ2 = 183)
Positive 62% (13/21) 38% (8/21) 0.012 44% (53/120) 56% (67/120) 0.038
Total
Negative 35% (208/593) 65% (385/593) (χ2 = 6.3) 34% (168/494) 66% (326/494) (χ2 = 4.3)

207

208 Fig 2. PCV distribution in trypanosome infected ( ) and non-infected ( ) animals by nPCR detection

6
 60

Number of observations
50

40

30

20

10

0
18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
PCV-values
209

210 4. Discussion
211 In this study, the prevalence of animal trypanosomiasis in tsetse infested areas of Somalia namely,
212 Afgoye and Jowhar districts was determined using microscopy (BCT) and PCR-based diagnostic techniques.
213 The overall prevalence of AAT in the present study is 3.4% by BCT and 19.5% by nPCR. This further
214 enforces the higher sensitivity of PCR methods over the microscopic detection of Trypanosoma spp. Similar
215 findings was reported by others (Angwech et al. 2015, N’Djetchi et al. 2017).
216 The nPCR result (19.5%) is in close agreement with the finding of Simwango et al. (2017) who
217 reported prevalence of 17.2% at Maasai Steppe, Northern Tanzania. However, our finding is lower than that
218 reported in Sudan (57.7%) by Osman et al. (2016) and Uganda (41%) by Angwech et al. (2015) but it is
219 higher than that reported by Laohasinnarong et al (2015) in Zambia (7.5%). Trypanosome evansi followed by
220 T. vivax are the predominant Trypansoma spp. in the study area as compared to other species. This may be due
221 to the camel grazing in the area and the presence of other biting flies which accelerate mechanical
222 transmission of the parasite.
223 The findings from this study have also shown a significant association between districts and
224 prevalence of trypanosome infections. This variation might be due to the differences in community practices
225 for trypanosomiasis and its vectors control. A higher prevalence of trypanosomiasis were recorded in cattle
226 (31.7%) compared to goats (18.4%) and sheep (8.7%). Similar results were reported by N’Djetchi et al (2017).
227 This may be contributed by the host susceptibility to the trypanosome infections and the skin coat suitability
228 for fly feeding as well.
229 The presence of T. simiae and T. godfreyi detected in the present study might be linked to the presence
230 of wild pigs (like Phacochoerus africanus [warthog], Potamochoerus larvatus somaliensis [bushpig]) in the
231 surveyed districts. None of sheep samples from both districts were indicated T. simiae infection. In contrast to
232 our study, Ng'ayo et al. (2005) indicated that sheep are naturally infected with T. simiae. These findings show
233 a research gap on the epizootiology of T. simiae infection in sheep.
234 Although the current study did not find T. b. rhodesiense in animals, the presence of potential vectors
235 and uncontrolled borders with infected neighbouring countries (Baker et al. 1970, Rutto and Karuga 2009)
236 might lead to introduction of the HAT parasite to the country.
237

238 5. Conclusion
239 The present study has shown that animal trypanosomiasis were prevalent in the studied areas despite
240 the ICRC’s T & T control projects. To the best of our knowledge, this is the first report of using molecular
241 (PCR) technique for the detection of Trypanosoma spp. in cattle and small ruminant from Somalia. Moreover,
242 T. godfreyi is the first time to be recorded in Somalia. Further large-scale studies and sustainable control
243 programmes of Trypanosoma spp. and its vectors are needed in the country.
7
244

245 Acknowledgments
246 The authors would like to thank Hassan Hussein and Abdiwali Mohamud at ARTC lab, Abrar University for
247 their help during blood samples collection and kind cooperation of animal owners who participated in this
248 study. We also acknowledge Makerere University for donation of T. b. rhodesiense DNA used as a positive
249 control in this study.

250 Authors Contributions

251 Conceptualization: Ahmed A. Hassan-Kadle, Abdalla M. Ibrahim and Rafael F. C. Vieira.
252 Data curation: Ahmed A. Hassan-Kadle, Abdalla M. Ibrahim.
253 Formal analysis: Ahmed A. Hassan-Kadle, Abdalla M. Ibrahim, Abdulkarim A. Yusuf.
254 Funding Acquisition: Ahmed A. Hassan-Kadle, Abdalla M. Ibrahim, Abdulkarim A. Yusuf.
255 Investigation: Ahmed A. Hassan-Kadle, Abdalla M. Ibrahim, Hamisi S. Nyingilili, Abdulkarim A. Yusuf.
256 Methodology: Ahmed A. Hassan-Kadle, Abdalla M. Ibrahim, Hamisi S. Nyingilili, Rafael F. C. Vieira.
257 Project administration: Ahmed A. Hassan-Kadle, Abdulkarim A. Yusuf and Rafael F. C. Vieira.
258 Resources: Ahmed A. Hassan-Kadle, Abdalla M. Ibrahim, Hamisi S. Nyingilili, Abdulkarim A. Yusuf.
259 Supervision: Ahmed A. Hassan-Kadle, Abdalla M. Ibrahim and Rafael F. C. Vieira.
260 Writing – original draft: Ahmed A. Hassan-Kadle.
261 Writing – review & editing: Ahmed A. Hassan-Kadle, Abdalla M. Ibrahim, Hamisi S. Nyingilili,
262 Abdulkarim A. Yusuf, Rafael F. C. Vieira.
263

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267 Emphasis to its Importance for “Guffar” Management and Control. Sud. J. Sci. Tech. 16. Pp. 69-76.
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