Infection, Genetics and Evolution 18 (2013) 6673

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Infection, Genetics and Evolution

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Identication and genetic characterization of Trypanosoma congolense

in domestic animals of Fontem in the South-West region of Cameroon
Gustave Simo a, , Pythagore Fogue Sobgwi a, Guy Roger Njitchouang b, Flobert Njiokou b,
Jules Roger Kuiate a, Gerard Cuny c, Tazoacha Asonganyi d
a
Department of Biochemistry, Faculty of Science, University of Dschang, Cameroon
b
Faculty of Science, University of Yaound 1, Cameroon
c
Institut de Recherche pour le Dveloppement, Unit Mixte de Recherche 177 IRD-CIRAD, Campus International de Baillarguet, TA A17/G, 34398 Montpellier Cedex 5, France
d
Faculty of Medicine and Biomedical Science, University of Yaound 1, Cameroon

a r t i c l e i n f o ab s t r a c t

Article history: To understand the circulation and the spread of Trypanosoma congolense genotypes in animals of Fontem
Received 11 February 2013 in the southwest region of Cameroon, T. congolense forest and T. congolense savannah were investigated in
Received in revised form 30 March 2013 397 domestic animals in eight villages. Out of the 397 domestic animals, 86 (21.7%) were found infected
Accepted 16 April 2013
by trypanosomes, using the capillary tube centrifugation test. The PCR with specic primers identied
Available online 25 April 2013
163 (41.1%) and 81 (20.4%) animals infected by T. congolense forest and T. congolense savannah, respec-
tively; showing for the rst time the circulation of T. congolense savannah in the Fontem region. No infec-
Keywords:
tion with T. congolense savannah was found in pigs whereas goats and sheep were infected by T.
Trypanosoma congolense
congolense forest and/or T. congolense savannah. The prevalence of trypanosomes varied signicantly
Microsatellite
Genotypes amongst villages and animal species. The genotyping of T. congolense forest positive samples using micro-
Animal African trypanosomiasis satellites markers showed that multiple genotypes occurred in 27.2% (44/163) of animals sampled,
whereas single genotypes were found in 73.8% (119/163) of samples. Some alleles were found in all ani-
mal species as well as in all villages and were responsible for major genotypes, whereas others (rare
alleles) were identied only in some animals of few villages. These rare alleles were characteristic of spe-
cic genotypes, assimilated to minor genotypes which can be spread in the region through tsetse ies.
The microsatellite markers show a low genetic variability and an absence of sub-structuration within
T. congolense forest. The analysis of the microsatellite data revealed a predominant clonal reproduction
within T. congolense forest. Pigs were the animal species with the highest number of different genotypes
of T. congolense forest. They seem to play an important epidemiological role in the propagation and spread
of different genotypes of T. congolense.
2013 Elsevier B.V. All rights reserved.

1. Introduction Most studies on African trypanosomes, especially the genetic

Animal African trypanosomiasis (AAT) also called nagana in cat-
tle is caused by Trypanosoma congolense (T. congolense), Trypano-
soma vivax (T. vivax) and Trypanosoma brucei (T. brucei) species.
Most of these trypanosomes are transmitted by the tsetse y
(genus Glossina). AAT constitutes one of the major constraints to
livestock production in 37 countries of sub-Saharan Africa (Swal-
low, 2000). This disease has been shown to be of high morbidity
and mortality in sub-Saharan livestock. It is estimated that control-
ling AAT would benet the agricultural industry by US$1300 mil-
lion per annum (Shaw, 2004). Because of nagana, stock farming
is very difcult within the tsetse belt of Africa (Steverding, 2008).
Corresponding author. Address: Department of Biochemistry, Faculty of science,
University of Dschang, P.O. Box 67, Dschang, Cameroon. Tel.: +237 94035497.
E-mail address: gsimoca@yahoo.fr (G. Simo).
characterization of trypanosomes, have been focused on T. brucei
(Nkinin et al., 2002; Simo et al., 2005, 2008, 2010), whereas only
few studies have been done with other African trypanosomes
including T. congolense. However, T. congolense is one of the major
causative agents of livestock disease. In wild animals, these para-
sites cause relatively mild infections while in domestic animals,
they might cause a severe, often fatal disease. Investigations
undertaken to understand the impact of T. congolense in the epide-
miology of animal trypanosomiaisis have shown that the highly
virulent strains of T. congolense cause an acute disease with high
mortality whereas other strains (with low virulence) result in a
chronic mild infection. Therefore, the differences in the pathologi-
cal and epidemiological evolutions of AAT may result from differ-
ent trypanosome strains that infect animals. In addition, the
variety of strains encountered in a T. congolense population may
determine the impact of the disease in a particular zone (Masumu

1567-1348/$ - see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.meegid.2013.04.019
 G. Simo et al. / Infection, Genetics and Evolution 18 (2013) 6673
et al., 2006). For instance, the experimental infections have shown
that the Kili type of T. congolense did not produce a patent infec-
tion while T. congolense savannah and forest types were both
chronic and caused mostly sub-patent parasitaemias (McNamara
et al., 1994).
Currently, considerable data has been generated on the preva-
lence, the localization and the distribution of animal trypanosomes
in Cameroon (Njiokou et al., 2004; Simo et al., 2006; Nimpaye et al.,
2011). This data enabled us to understand some epidemiological
aspects of the disease notably the transmission and spread of the
disease. However, many important basic questions related to the
biology, genetic diversity and population genetics of T. congolense
are still not well understood. Answering these questions will be
fundamental for our knowledge on the trypanosome biology,
diversity and evolution of these parasites. For instance, the genetic
characterization of trypanosomes may enable us rene our knowl-
edge on the genetic diversity of T. congolense populations as well as
the spread and distribution of specic strains or genotypes.
Previous data on T. congolense population diversity come from
isoenzyme electrophoresis (Gashumba et al., 1988; Young and
Godfrey, 1983). Used to analyze groups of eld isolates, this bio-
chemical tool has shown a genetic heterogeneity within T. congo-
lense with the subdivision of the species into three genetically
distinct subgroups designated as: Savannah, Kili and Forest
(Gashumba et al., 1988; Young and Godfrey, 1983). At the genetic
level, isoenzyme electrophoresis has shown high levels of hetero-
zygosity, an overrepresentation of identical genotypes and linkage
disequilibrium (Tibayrenc et al., 1991). This led to the conclusion
that this species was predominantly clonal. Thereafter, microsatel-
lite markers specic for T. congolense were developed from the gen-
ome of this parasite (Morrison et al., 2009). The analysis of the
genotyping data of the Gambian T. congolense isolates from live-
stock strongly supported the occurrence of mating in this
trypanosome.
The higher sensitivity and specicity of the microsatellite mark-
ers enabled their use for the genetic characterization of T. brucei di-
rectly from biological samples including tsetse midguts, blood and
cerebrospinal uid (Kabore et al., 2011; Simo et al., 2011, 2012;
Truc et al., 2012). In human African trypanosomiasis, microsatellite
markers provided considerable data that enabled us to understand
some biological and epidemiological aspects of trypanosomiasis as
well as the population genetics of T. brucei. For T. congolense, seven
microsatellite markers were used to investigate the mating system
(Morrison et al., 2009).
In this study, our objective was to identify T. congolense circulat-
ing in domestic animals of the Fontem region of the southwest re-
gion of Cameroon. Thereafter, a molecular characterization of these
parasites was performed with microsatellite markers in order to
evaluate the genetic diversity of T. congolense forest, distribution
and spread of genotypes in villages and domestic animal species
of the southwest region of Cameroon.
2. Materials and methods
2.1. Study area
The Fontem region (05400 1200 N, 09550 3300 E) is located in the
Lebialem, Manyu and Koupe-Manengouba divisions of Cameroon.
This region is characterized by a tropical humid climate with var-
ied topography of hills and valleys through which several high
speed rivers ow. The main population activities are subsistence
agriculture, palm oil extraction, animal husbandry and small scale
poultry farming. The dense population of humans, domestic ani-
mals (dogs, pigs, sheep, and goats) and tsetse ies are found scat-
tered in the pre-forest/forest vegetation of the valleys and hills.
67
Glossina palpalis palpalis is the main vector of trypanosomes in this
locality (Morlais et al., 1998). Previous studies on animal and hu-
man African trypanosomiasis revealed several species and subspe-
cies of trypanosomes in various domestic animal species of this
region (Nkinin et al., 2002; Simo et al., 2006; Nimpaye et al., 2011).
2.2. Blood collection and parasitological analysis
The domestic animals analyzed in this study were sampled dur-
ing two eld surveys in the Fontem region: the rst survey was
performed in July 2006 and the second in June 2007. These surveys
were carried out in eight villages: Besali, Bechati, Folepi, Agong,
Nsoko, Fossung, Menji and Azi (Fig. 1). Before each survey, the
objective of the study was rst explained to inhabitants and local
authorities of the villages. After obtaining their approval, the
inhabitants were asked to restrain and/or keep their domestic ani-
mals. In each village, all domestic animals that had spent at least
3 months in the study zone were selected. From each animal, about
5 ml of blood was collected into EDTA coated tubes. This blood was
collected from jugular vein in goats, pigs and sheep and from sa-
phena vein in dogs. All the pigs and dogs sampled in this study
were of local breed, originating from many different breeds. The
sheep and goats were dwarf breeds (Djallonke west-African dwarf
for sheep and Guinea goat), which are known to be trypanotoler-
ant. Some pigs were kept in pigsties whereas other animals sam-
pled were allowed to move freely around the villages.
The capillary tube centrifugation test, as described by Woo
(1970), was performed on each sample to investigate the presence
of trypanosomes. The remaining blood samples were conserved in
an icebox and transported to the laboratory, where they were
stored at 4 C before DNA extraction.
2.3. DNA extraction
The DNA was extracted from 1 ml of blood using the DNA kit
DNeasy Tissue kit (Qiagen) as described by Simo et al. (2012).
Briey, 1 ml of blood was mixed with equal volume of nuclease
free water. The mixture was homogenized for 10 min and thereaf-
ter, centrifuged at 14,000 rpm for 10 min. After this centrifugation,
the supernatant was discarded and the pellet was resuspended in
200 ll of PBS buffer. The suspension was used for DNA extraction
following the instructions of the manufacturer. The DNA extract
was used directly for PCR or stored at 20 C to be used later.
2.4. Identication of T. congolense savannah and T. congolense forest
The identication of different types of T. congolense was per-
formed with TCF1/2 (Masiga et al., 1992) and TCS1/2 (Majiwa
et al., 1994) specic primers for T. congolense forest and T. congo-
lense savannah, respectively. For each PCR reaction, the amplica-
tions were performed in a nal volume of 25 ll containing 5 ll of
DNA extract, 1.5 mM of MgCl2 (nal concentration), 20 pmol of
each primer, 200 mM of each dNTP and 1 unit of Taq DNA polymer-
ase. The amplication cycles contained one denaturing step at
94 C for 5 min followed by 40 amplication cycles. Each cycle
was constituted by a denaturation step at 94 C for 30 s, an anneal-
ing step at 60 C for 30 s, and an extension step at 72 C for 1 min.
This was followed by a nal extension at 72 C for 10 min.
At the end of each amplication reaction, 10 ll of each ampli-
ed product was resolved on 1.5% agarose gel containing ethidium
bromide (0.3 mg/ml). The gels were observed under UV light, and
then photographed. All samples showing a specic DNA fragment
for T. congolense forest were selected for subsequent genetic char-
acterization by microsatellite markers.