Parasitological, serological and molecular survey of camel trypanosomiasis in Somalia

Abstract

Background:
Camel trypanosomiasis or surra is of great concern in Somalia, since the country possesses the largest one-humped camel (Camelus dromedarius) population in the world. Civil war in Somalia has resulted in the destruction of educational, research, economic and social structures, making the country scores very low for most humanitarian indicators. Previous studies on detection of Trypanosoma species in Somali camels have only been performed during the 1990s using standard trypanosome detection methods (STDM). Considering the lack of state-of-the-art knowledge on camel trypanosomiasis in Somalia, the present study aimed to assess the prevalence of Trypanosoma spp. in three districts of Somalia.

Methods:
A total of 182 blood samples from C. dromedarius from nomadic and dairy farms were evaluated using STDM, serological (CATT/T. evansi) and molecular (ITS1-PCR) methods.

Results:
All samples were negative for Trypanosoma spp. by STDM. A total of 125/182 (68.7%, 95% CI: 61.4-75.3%) camels were seropositive for T. evansi by CATT/T. evansi. Camels reared in nomadic system were more likely to be seropositive for T. evansi than those under dairy production system (OR: 5.6, 95% CI: 2.1-15.2, P = 0.0001). Five out of 182 (2.7%, 95% CI: 0.9-6.3%) camels tested positive for Trypanosoma sp. by ITS1-PCR. Sequencing of the ITS1 region of the Trypanosoma species detected herein revealed that camels were infected with T. evansi and T. simiae.

Conclusions:
Trypanosoma evansi is highly prevalent in camels from the Banadir region of Somalia, particularly in nomadic herds. To our knowledge, this is the first study to confirm infections with T. evansi and T. simiae in Somali camels through DNA sequencing. Our data highlight the need for implementation of adequate control measures aiming to reduce the impact on camel production in the country.

Full text

Hassan‑Kadleetal. Parasites Vectors (2019) 12:598
https://doi.org/10.1186/s13071‑019‑3853‑5
RESEARCH
Parasitological, serological andmolecular
survey ofcamel trypanosomiasis inSomalia
Ahmed A. Hassan‑Kadle1,2*† , Abdalla M. Ibrahim1†, Hamisi S. Nyingilili3, Abdulkarim A. Yusuf1,2,
Thállitha S. W. J. Vieira2 and Rafael F. C. Vieira2,4*
Abstract
Background: Camel trypanosomiasis or surra is of great concern in Somalia, since the country possesses the largest
one‑humped camel (Camelus dromedarius) population in the world. Civil war in Somalia has resulted in the destruc‑
tion of educational, research, economic and social structures, making the country scores very low for most humani‑
tarian indicators. Previous studies on detection of Trypanosoma species in Somali camels have only been performed
during the 1990s using standard trypanosome detection methods (STDM). Considering the lack of state‑of‑the‑art
knowledge on camel trypanosomiasis in Somalia, the present study aimed to assess the prevalence of Trypanosoma
spp. in three districts of Somalia.
Methods: A total of 182 blood samples from C. dromedarius from nomadic and dairy farms were evaluated using
STDM, serological (CATT/T. evansi) and molecular (ITS1‑PCR) methods.
Results: All samples were negative for Trypanosoma spp. by STDM. A total of 125/182 (68.7%, 95% CI: 61.4–75.3%)
camels were seropositive for T. evansi by CATT/T. evansi. Camels reared in nomadic system were more likely to be sero‑
positive for T. evansi than those under dairy production system (OR: 5.6, 95% CI: 2.1–15.2, P = 0.0001). Five out of 182
(2.7%, 95% CI: 0.9–6.3%) camels tested positive for Trypanosoma sp. by ITS1‑PCR. Sequencing of the ITS1 region of the
Trypanosoma species detected herein revealed that camels were infected with T. evansi and T. simiae.
Conclusions: Trypanosoma evansi is highly prevalent in camels from the Banadir region of Somalia, particularly in
nomadic herds. To our knowledge, this is the first study to confirm infections with T. evansi and T. simiae in Somali
camels through DNA sequencing. Our data highlight the need for implementation of adequate control measures aim‑
ing to reduce the impact on camel production in the country.
Keywords: CATT/T. evansi, Dromedary, ITS1‑PCR, Trypanosoma evansi, Trypanosoma simiae
© The Author(s) 2019. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and
the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material
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main/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Background
Trypanosomiasis are vector-borne diseases (VBD) that
causes noticeable economic losses [1–3] and affects
the development of both livestock and human health in
Africa [4]. In Somalia, camel trypanosomiasis or surra is
of great concern since the country possesses the largest
one-humped camel (Camelus dromedarius) population
in the world, estimated at nearly 8,000,000 heads [5–7].
e economic importance of camels for Somalia is due
to their role as a food source, as currency, as a means of
transporting milk and water as well as an indicator of
social issues [7]. Camels are uniquely adapted to survive
and produce under extreme arid and semi-arid condi-
tions of Somalia [7], with the majority of animals kept by
nomadic pastoralists in the country [6, 7].
Open Access
Parasites & Vectors
*Correspondence: akadle@abrar.edu.so; rvieira@ufpr.br
†Ahmed A. Hassan‑Kadle and Abdalla M. Ibrahim contributed equally to
this work
1 Abrar Research and Training Centre, Abrar University, Mogadishu,
Somalia
2 Department of Veterinary Medicine, Universidade Federal do Paraná,
Curitiba, PR, Brazil
Full list of author information is available at the end of the article
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Page 2 of 6
Hassan‑Kadleetal. Parasites Vectors (2019) 12:598
Civil war in Somalia has resulted in the destruction of
educational, research, economic and social structures,
making the country score very low for most humanitar-
ian indicators [8]. Currently, Somali communities and
their livestock are experiencing a famine and suffer-
ing from preventable diseases, due to geographical and
political isolation and lack of state-of-the-art knowledge.
Recently, after some security and political settlement
in the country, camels are kept around urban areas as a
semi-intensive dairy farming system.
Worldwide, camels may be affected by tsetse-transmit-
ted Trypanosoma species, including T. simiae, T. brucei,
T. congolense and T. vivax [9, 10]. In Somalia, previous
studies on detection of Trypanosoma species were per-
formed during the 1990s and reported T. evansi preva-
lence rates ranging from 1.7% to 56.4% in camels [7, 11].
Additionally, T. simiae [9], T. congolense and T. brucei
have also been detected in Somali camels by standard
trypanosome detection methods (STDM) [11].
Clinical signs of trypanosomiasis may be absent in
camels, and thus, laboratory diagnosis should be car-
ried out for confirmation of infection. Several methods
with varying degrees of sensitivity and specificity may
be used for the diagnosis of trypanosomiasis. Standard
trypanosome detection methods, such as microscopical
examination of fresh or stained blood-smears, has been
historically used in the identification of Trypanosoma
spp. Unfortunately, this technique lacks sensitivity and
specificity. A serological assay, the card agglutination
test for T. evansi (CATT/T. evansi) is a rapid diagnostic
test and currently recommended by the World Organiza-
tion for Animal Health [2, 12]. Additionally, molecular
analysis targeting the internal transcribed spacer 1 (ITS1)
region provides multi-species-specific detection of trypa-
nosomes in a single PCR [13], and has been used in epi-
demiological studies.
Although the National Tsetse and Trypanosomiasis
Control Project (NTTCP) was established in the 1980s
in Somalia, no control measures have been implemented
to date. Accordingly, considering the lack of state-of-
the-art knowledge on camel trypanosomiasis in Soma-
lia, the present study aimed to assess the prevalence of
camel trypanosomiasis in three districts of Somalia using
STDM, serological (CATT/T. evansi) and molecular
(ITS1-PCR) methods.
Methods
Study area
Banadir region is one of the eighteen regions of the Fed-
eral Republic of Somalia. e region itself is coextensive
with Mogadishu city, the capital of the country. It con-
sists of 17 districts, and three of them were included in
this study: Kahda (2° 4′ 4.17″ N, 45° 14′ 16.16″ E), Daynile
(2° 4′ 24.61″ N 45° 16′ 48″ E) and Yaqshid (2° 4′ 3.97″ N,
45° 21′ 35.9″ E). ese districts are the main camel rear-
ing areas in the investigated region.
Study animals andblood sampling
From December 2015 to March 2016, which represents
the dry season in Somalia, a total of 182 C. dromedarius
(176 females and 6 males) ≥ 2 years-old from nomadic
(n = 49) and dairy (n = 133) farms in the Kahda (n = 72),
Daynile (n = 87) and Yaqshid (n = 23) districts were ran-
domly evaluated. Blood samples were collected by jugu-
lar venipuncture. ree millilitres were placed into tubes
without anticoagulant and kept at room temperature
(25 °C) until visible clot retraction; the samples were
then centrifuged at 1500× g for 5min, serum separated
and kept at − 20 °C for serological studies. One ml was
placed into EDTA tubes for packed cell volume (PCV)
measurement, microscopical detection of trypanosomes
and preparation of blood spots on filter paper (Whatman
no.4, Whatman, Springfield Mill, United Kingdom) for
PCR analysis. A PCV of 0.26 l/l or less was used as an
indicator of anaemia [14].
Parasitological diagnosis ofTrypanosoma spp.
All camel blood samples were evaluated for the presence
of Trypanosoma spp. by STDM. Briefly, a drop of fresh
whole blood (after gentle mixing) was placed on a clean
microscope slide, covered with coverslip and examined
for the motile parasites, as previously described [15].
Giemsa-stained thin blood and buffy coat smears were
also examined for the presence of Trypanosoma spp., as
described elsewhere [2].
Detection ofT. evansi antibodies bycard agglutination test
(CAT T/T. evansi)
Camel serum samples were tested for the presence of
T. evansi antibodies using the card agglutination test
(CATT/T. evansi) [16], according to the manufacturer’s
instructions (Institute of Tropical Medicine, Antwerp,
Belgium).
DNA extraction andPCR forTrypanosoma spp.
Genomic DNA was extracted from all 182 dried blood
spots by Chelex-100 (Sigma-Aldrich, St. Louis, USA), as
previously described [17]. e DNA samples were evalu-
ated by a PCR assay targeting the ITS1 region of Trypa-
nosoma species using previously described primers [18].
e PCR amplifications were performed in a total reac-
tion volume of 25μl containing 0.5μl of 10 pM of each
primer, 12.5μl of 2× master mix (New England BioLabs,
Ipswich, MA, USA), 9.5μl of PCR water and 2μl of each
DNA template. PCR amplifications were performed with
a thermal cycler (GeneAmp® PCR System 9700, Applied
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

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Hassan‑Kadleetal. Parasites Vectors (2019) 12:598
Biosystems®, Foster City, CA, USA). e amplification
conditions used included an initial denaturation at 94°C
for 30 s, followed by 30 cycles of 94°C for 30 s, 58°C for
40 s, 68°C for 1min, with a final extension step at 68°C
for 5min and cooling at 4°C. Nuclease-free water and a
T. evansi-positive sample were used as negative and posi-
tive control, respectively, in all PCR runs. e amplified
PCR products were analysed by electrophoresis in a 1.5%
agarose gel at 100–120 V for 60min. Quick Loading 100
bp DNA ladder (New England BioLabs) was included
on each gel, stained with ethidium bromide, and finally
visualized under ultraviolet (UV) illuminator (UVITEC™,
Cambridge, UK).
Sequencing andphylogenetic analysis
Amplicons (~ 400bp) obtained from two Trypanosoma-
positive samples were sequenced in both directions by
the Sanger method and were assembled using Geneious
Prime® 2019.1. Consensus sequences were subjected to
BLASTn analysis [19] for determining the identity with
the sequences deposited in the GenBank database.
e Trypanosoma ITS1 region sequences (GenBank:
MH885470, MH885471) were aligned with sequences
from GenBank using ClustalW [20] and alignments were
improved using GUIDANCE2 [21]. e best-fit model of
nucleotide substitution was determined using jModeltest
v.2.1.10 [22] and was set as F81+G in the maximum like-
lihood (ML) phylogenetic estimation on the CIPRES Sci-
ence Gateway [23], including 1000 bootstrap replicates.
e resulting tree was visualized using FigTree software
version 1.4.3 [24] and the final layout was rendered using
Inkscape version 0.92.3 [25].
Data management andanalysis
e PCV data were not normally distributed (Shap-
iro–Wilk normality test, W = 0.98, P = 0.018). ere-
fore, a non-parametric Mann–Whitney test was
used to compare the PCV concentration between
Trypanosoma-infected and non-infected camels. Either
Chi-square or Fisher’s exact test was used to assess asso-
ciation of the individual variables (district and produc-
tion system) with Trypanosoma spp. infection. Odds
ratio (OR), 95% confidence intervals (95% CI) and P-val-
ues were calculated, and results were considered signifi-
cant when P < 0.05. Data were compiled and analysed by
Statistical Package for Social Sciences (SPSS) version 25
(IBM Corp., Armonk, NY, USA).
Results
All samples were negative for Trypanosoma spp. by
STDM. A total of 125/182 (68.7%, 95% CI: 61.4–75.3%)
camels were seropositive for T. evansi by CATT/T. evansi.
Camels reared in the nomadic system were more likely
to be seropositive to T. evansi than those reared under
the dairy production system (OR: 5.6, 95% CI: 2.1–15.2,
P = 0.0001). No associations between seropositivity for T.
evansi and the three districts of Somalia evaluated were
found (P > 0.05). e prevalence of Trypanosoma sp. for
each variable evaluated is summarized in Table1.
Five out of 182 (2.7%, 95% CI: 0.9–6.3%) camels tested
positive for Trypanosoma spp. by ITS1-PCR. Concord-
ant results for Trypanosoma spp. presence determined
by CATT/T. evansi and ITS1-PCR were found in three of
182 camels (1.6%, 95% CI: 0.3–4.7%).
e mean PCV concentration for camels was 0.27 l/l. A
total of 61/182 (33.5%, 95% CI: 26.7–40.9%) camels were
anaemic. No statistical difference (U = 2944, Z = − 1.89,
P = 0.059) was found in mean PCV between Trypano-
soma-seropositive (0.27 l/l) and Trypanosoma-seronega-
tive camels (0.28 l/l). Association between Trypanosoma
infection and anaemia was not found (χ2 = 1.93, df = 1,
P = 0.165).
Five Trypanosoma-positive samples were sequenced;
however, only two sequences yielded consistent
data. One Trypanosoma-positive sample (GenBank:
MH885471) sequenced showed 99.78% (460/461 bp)
Table 1 Prevalence of camel trypanosomiasis within each variable studied
Abbreviations: +, number of positive animals; n, number of samples; 95% CI, 95% condence interval; OR, odds ratio
Variable CATT /T. evansi ITS1‑PCR
+/nPrevalence (%) (95% CI) P‑value OR (95% CI) +/nPrevalence (%) (95% CI) P‑value OR (95% CI)
Production system
Nomadic 44/49 89.8 (77.8–96.6) 0.0001 (χ2 = 13.9) 5.6 (2.1–15.2) 2/49 4.1 (0.5–14) 0.408 (χ2 = 0.45) 1.8 (0.3–11.4)
Dairy 81/133 60.9 (52.1–69.2) 3/133 2.3 (0.5–6.5)
District
Daynile 62/87 71.3 (60.6–80.5) 0.242 (χ2 = 1.38) 1.5 (0.8–2.9) 2/87 2.3 (0.3–8.1) 0.502 (χ2 = 0.45) 0.5 (0.09–3.33)
Yaqshid 18/23 78.3 (56.3–92.5) 0.164 (χ2 = 1.94) 2.2 (0.7–6.5) 0/23 0.0 (0.0–14.8) 0.989 (χ2 = 0.32) 0.0
Kahda 45/72 62.5 (50.3–73.6) 3/72 4.2 (0.9–8.8)
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Hassan‑Kadleetal. Parasites Vectors (2019) 12:598
identity to T. evansi ITS1 region sequences detected in
camels from Iran (GenBank: KX898422, KX898423). e
other sequence obtained (GenBank: MH885470) showed
99.25% (398/401 bp) and 98% (393/401 bp) identity with
T. simiae ITS1 region sequence from warthogs (Phaco-
choerus africanus) of Tanzania and Zambia, respectively
(GenBank: JN673387 and JN673386). e phylogenetic
tree based on sequences of the ITS1 region indicated that
T. evansi obtained herein was closely related to T. evansi
detected in camels from Iran, whereas T. simiae detected
in the present study was closely related to T. simiae
detected in warthog from Zambia (Fig.1).
Discussion
To the author’s knowledge, this is the first study to
combine STDM, serological and molecular detection
of Trypanosoma and assess these results for potential
associations with epidemiological data collected from
camels in Somalia. Herein, overall 69.8% camels from the
Banadir region of Somalia were positive for Trypanosoma
spp. Interestingly, Trypanosoma prevalence found herein
was higher than previous studies performed in camels
from Somalia which have shown prevalence rates rang-
ing from 1.7% to 56.4% by STDM [7, 11] and complement
fixation test [7]. Differences among the prevalence of
Trypanosoma may be explained by the camel population
and management, diagnostic test used, and tsetse sea-
sonal dynamics (rainy vs dry season).
Sequencing of the ITS1 region of the Trypanosoma
species detected herein revealed that camels were
infected with T. evansi and T. simiae (Fig.1). Although
previous studies in Somalia have reported T. evansi, T.
simiae, T. brucei and T. congolense infecting camels by
STDM, the present study is the first to confirm infections
with Trypanosoma spp. in camels in this country by DNA
sequencing.
0.8
Trypanosoma congolense from Hyaena (JN673388) – Tanzania
Trypanosoma evansi from Camel (JN896755) – Iran
Trypanosoma simiae from Warthog (JN673386) – Zambia
Trypanosoma evansi from Camel (KX898423) – Iran
Trypanosoma evansi from Camel (KX898422) – Iran
Trypanosoma evansi from Camel (FJ712716) – China
Trypanosoma simiae from Camel (MH885470) – Somalia
Trypanosoma simiae from Warthog (JN673387) – Tanzania
Trypanosoma evansi from Camel (JN896754) – Iran
Trypanosoma godfreyi from Warthog (JN673385) – Ta nzania
Trypanosoma evansi from Camel (MH885471) – Somalia
Trypanosoma simiae from Warthog (JN673382) – Tanzania
Trypanosoma godfreyi from Warthog (JN673384) – Zambia
Trypanosoma congolense from Lion (JN673389) – Tanzania
Leishmania sp. (EF524071)
100
100
69
49
100
100
78
59
98
98
100
100
53
Fig. 1 Phylogenetic relationships of Trypanosoma spp. evaluated in camels based on ITS1 region sequence with selected sequences from GenBank
(Accession numbers in the figure). The ITS1 tree was rooted with Leishmania sp. (GenBank: EF524071)
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Hassan‑Kadleetal. Parasites Vectors (2019) 12:598
In the present study, all animals were negative for
Trypanosoma by STDM, while the molecular and sero-
logical prevalence were 2.7% and 68.7%, respectively.
Combining CATT/T. evansi and ITS1-PCR has increased
the prevalence of Trypanosoma to 69.8%, corroborating
with previous studies suggesting that improved sensitiv-
ity and specificity for detection of VBD pathogens can
be achieved using different diagnostic methods [26]. e
difference between the ratio of ITS1-PCR and parasito-
logical method found herein may be explained by low
parasitaemia which is typical for the chronic phase of
infection [27]; this is also supported by the high sero-
prevalence observed indicating that a large proportion of
camels are exposed to the parasite.
Herein, the T. evansi seroprevalence rate was signifi-
cantly higher in nomadic camels as compared to dairy
farm camels (P = 0.0001). Previous studies on Somali
camels have shown that animals living in riverine zones
of the country were more likely to acquire infection by
Trypanosoma species than those living in inland zones
[7]. e camels’ role in the subsistence sector is not pri-
marily for supply of meat and money, but mainly for
provision of milk [7]. Considering that nomadic herders
usually depend on traditional ethno-veterinary remedies
to treat and prevent diseases in their camels [28, 29], the
high T. evansi seroprevalence found herein highlights the
need for implementation of adequate control measures
aiming to reduce the impact of trypanosomes on camel
production in Somalia. On the other hand, dairy animals
evaluated in the present study were frequently examined
by veterinary practitioners and treated with Suramin
(data not shown), which may explain the low seropreva-
lence found.
Conclusions
Trypanosoma evansi is highly prevalent in camels from
the Banadir region of Somalia, particularly in nomadic
herds. To our knowledge, this is the first study to confirm
infections with T. evansi and T. simiae in Somali cam-
els by DNA sequencing. Our data highlight the need for
implementation of adequate control measures aiming to
reduce the impact of trypanosomes on camel production
in the country, which possesses the largest one humped
camel population in the world.
Abbreviations
ARTC : Abrar Research and Training Centre; AU: Abrar University; BLAST: basic
local alignment search tool; CATT : card agglutination test for trypanosomia‑
sis; CI: confidence interval; DNA: deoxyribonucleic acid; GOHi: Global One
Health Initiative; ITS: internal transcribed spacer; ML: maximum likelihood;
NTTCP: National Tsetse and Trypanosomiasis Control Project; OR: odds ratio;
PCR: polymerase chain reaction; PCV: packed cell volume; SPSS: statistical
package for social sciences; STDM: standard trypanosome detection methods;
UV: ultraviolet; VBD: vector‑borne diseases; VVBDI: Vector and Vector Borne
Diseases Institute.
Acknowledgements
The authors thank Dr. Ehab Mossaad, Sudan University of Science and
Technology, Sudan for providing CATT/T. evansi kits. We also thank the team
at VVBDI, Tanzania for their molecular services. The Brazilian National Council
of Scientific and Technological Development (CNPq) provided a fellowship of
research productivity (PQ) to Dr. Rafael Vieira.
Authors’ contributions
AAHK, AMI and RFCV designed the study. AAHK, AMI and AAY collected the
data. AAHK and AMI curated the data. AAHK, AMI, AAY, HSN, TSWJV and RFCV
carried out the methodology. AAHK, AMI, AAY and TSWJV performed the data
analysis. AAHK, AMI, HSN and RFCV drafted the manuscript. All authors read
and approved the final manuscript.
Funding
This study was financially supported by the Abrar University, Somalia, with Ref‑
erence number AURG02012015. The sponsor had no role in the design of the
study, collection, analysis and interpretation of data, writing the manuscript
and the decision to submit the article for publication.
Availability of data and materials
Data supporting the conclusions of this article are provided within the article.
Sequences were submitted to the GenBank database under the Accession
numbers MH885470 and MH885471 for Trypanosoma ITS1 region of T. simiae
and T. evansi, respectively.
Ethics approval and consent to participate
This study was approved by the ethical committee of Abrar University, Somalia
(Reference number AU/ARTC/EC/02/2015). All camel owners gave consent to
sample their animals.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1 Abrar Research and Training Centre, Abrar University, Mogadishu, Somalia.
2 Department of Veterinary Medicine, Universidade Federal do Paraná, Curitiba,
PR, Brazil. 3 Vector and Vector Borne Diseases Institute, Tanga, Tanzania. 4 Global
One Health Initiative (GOHi), The Ohio State University, Columbus, OH, USA.
Received: 30 July 2019 Accepted: 14 December 2019
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