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Opinion
African trypanosomes: celebrating
diversity
Emily R. Adams1, Patrick B. Hamilton2 and Wendy C. Gibson3
1
Koninklijk Instituut voor de Tropen (KIT) Biomedical Research, Amsterdam 1105 AZ, Netherlands
School of Biosciences, University of Exeter, UK, EX4 4PS
3
School of Biological Sciences, University of Bristol, UK, BS8 1UG
2
Recent advances in molecular identification techniques
and phylogenetic analysis have revealed the presence of
previously unidentified tsetse-transmitted trypanosomes in Africa. This is surprising in a comparatively
well-known group of pathogens that includes the causative agents of human and animal trypanosomiasis.
Despite levels of genetic divergence that warrant taxonomic recognition, only one of these new trypanosomes
has been named as a new species; the increased diversity is largely ignored or regarded as an inconvenient
complication. Yet, some of these trypanosomes have
demonstrated pathogenicity, whereas others are closely
related to known pathogens, and might share this trait.
We should first acknowledge that these novel trypanosomes exist and then take steps to investigate their host
range, pathogenicity to livestock and response to chemotherapy.
Molecular identification as a tool for discovering new
diversity
Over the last few years molecular identification and phylogenetic analysis have revealed the presence of several
previously unidentified trypanosome species carried by
tsetse flies in sub-Saharan Africa (Table 1) [1–5]. This is
surprising in such a notorious and well-studied group of
pathogens, among which are the parasites responsible for
sleeping sickness in humans and nagana in livestock.
These devastating diseases continue to have a major
impact on human and animal health across the vast
regions of Africa infested with tsetse flies. Despite levels
of genetic divergence that warrant taxonomic recognition,
only one of these novel trypanosomes has been named as a
new species, Trypanosoma godfreyi [1]. The availability of
isolates collected from the field allowed the full species
description of this trypanosome, including a morphometric
analysis of bloodstream forms and description of the complete developmental cycle in pigs and tsetse flies [1]. Even
so, T. godfreyi has not yet made it to standard texts,
although experiments have demonstrated that it is pathogenic to pigs [1]. Instead, only a handful of traditionally
acknowledged species are accepted (Table 2), and the
newly discovered diversity is either ignored or regarded
as an inconvenient complication. We propose here that
most of the novel trypanosomes are likely to be livestock
pathogens and that we should take steps to investigate
Corresponding author: Adams, E.R. ([email protected]).
their host range, pathogenicity and response to chemotherapy. Additionally, these new genotypes could serve as
valuable comparisons for the known pathogens and provide insights into their virulence and pathogenicity.
Why are novel trypanosomes being discovered now?
Traditionally, workers described many tsetse-transmitted
species on the basis of the morphology of bloodstream
forms, host range and pathogenicity, but over time taxonomic revisions have removed synonymous or poorly
characterized species, leaving only a few commonly recognized species [6] (Table 2). Now, however, the high discriminatory power of molecular techniques for
identification is revealing far greater levels of genetic
diversity than acknowledged by current nomenclature.
Similar problems are encountered in other groups of pathogenic protists, where there are insufficient distinguishing
morphological features for identification by microscopy
alone. Initially, it was difficult to gauge the level of divergence between different trypanosome genotypes, but the
increasing sophistication and accessibility of molecular
phylogenetic analysis allows the accurate phylogenetic
placement of any new trypanosome after sequencing just
a couple of genes. Indeed this has been so successful that
there are now a multitude of newly discovered trypanosomes jostling for recognition as new species (Table 1)
(Figure 1).
The pace of discovery of new genotypes has been
increased through the recent introduction of generic
(genus-specific) methods for identification of trypanosomes; these methods are able to detect and identify a
wide diversity of trypanosomes, rather than single species,
at the same time. Generic identification methods have the
potential to identify all known species and also detect novel
species. These methods are PCR-based, with primers
complementary to conserved regions that flank more variable sequences. For example, the internal transcribed
spacer (ITS) that separates the 18S and 5.8S ribosomal
RNA (rRNA) genes and varies both in sequence and length
among trypanosome species, is easily amplified using generic primers matching each of the conserved rRNA genes
[7,8]. The sequence of this region is known for a number of
trypanosome species as well as many other organisms,
making it straightforward to design generic primers that
match a diversity of trypanosome species, while avoiding
amplification of host DNA. Since the method is PCR-based,
samples with relatively few organisms will yield results,
1471-4922/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2010.03.003 Available online xxxxxx
1
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Trends in Parasitology Vol.xxx No.x
Table 1. Novel trypanosome genotypes described in the past 30 years.
Subgenus
Undetermined
Nannomonas
Duttonella
b
a
Trypanosome
T. brucei-like c
Vertebrate hosts
Unknown
T. congolense savannah
T. congolense forest
Broad range including ungulates
and carnivores
Recorded from pigs, goats, cattle, dogs.
T.
T.
T.
T.
T.
T.
T.
Recorded from cattle, sheep and goats
Only pigs infected experimentally
Unknown
Only pigs infected experimentally
Unknown
Unknown
Nyala antelope, and experimental infection in goats
congolense Kilifi
godfreyi
godfreyi-like c
simiae Tsavo
vivax A c
vivax B c
vivax C - Nyala
Known distribution
Coastal Tanzania
Tropical Africa
d
Riverine-forest biomes in
West and Central Africa
East Africa
Tropical Africa d
Coastal Tanzania
East Africa
Tanzania
Tanzania
Mozambique
Reference
[2]
[10,11]
[10,11]
[11,13,14]
[1]
[3,8]
[5,15]
[4]
[4]
[31]
a
Several of the trypanosomes listed have been recorded from tsetse hosts only and the vertebrate host range is unknown.
The transmission cycle of the T. brucei-like trypanosome is incompletely known, and hence it cannot be assigned to one of the existing subgenera, which are defined on the
developmental cycle within the vector. It is sufficiently genetically divergent to represent a new subgenus.
c
Trypanosomes identified solely by DNA sequence data currently.
d
Recorded from many regions of sub-Saharan Africa where tsetse flies are present.
b
and sensitivity can be increased further by using a nested
PCR design.
The 18S and 28S rRNA genes lend themselves to the
design of simple generic PCR tests because they are composed of alternating conserved and variable domains,
allowing the variable domains to be amplified using primers in the flanking conserved regions. This principle was
incorporated into the high throughput method of fluorescent fragment length barcoding (FFLB) [2]. FFLB combines accuracy with speed by analyzing multiple variable
regions of the 18S and 28S rRNA genes using fluorescentlylabeled PCR fragments size-fractionated on an automated
sequencer. The use of several variable regions in FFLB,
compared to only one in ITS-PCR, increases the discriminatory power.
Sequence analysis provides the ultimate information for
genetic identification, and allows new trypanosomes to be
placed within the phylogenetic tree. Sequences are a permanent record of identity making it easy to compare new
sequences with those from reference isolates held in public
databases. For example, there are now well over 100
trypanosome 18S rRNA gene sequences on the GENBANK
database (www.ncbi.nlm.nih.gov) for comparison with any
new sequence. The approximately 800 bp D7-D8 region is
the most variable region and sometimes described as
the barcoding gene. To complement the rDNA data with
Table 2. Traditionally recognized species
Subgenus
Trypanozoon
Nannomonas
Duttonella
Pycnomonas
Developmental sites
in tsetse
Midgut and salivary
glands
Midgut and proboscis
Proboscis and cibarial
pump
Midgut, salivary glands
and proboscis
a
Species
Trypanosoma brucei
sensu lato
T. congolense,
T. simiae
T. vivax,
T. uniforme
T. suis
a
The traditionally recognized species of African trypanosomes transmitted by tsetse
via the salivarian route [6]. T. (Pycnomonas) suis is included for completeness,
although it has not been recorded for over 30 years, and T. (Duttonella) uniforme is
rarely reported. All these species are known pathogens of humans (T. brucei) or
livestock (all species listed).
2
information derived from a protein-coding gene, the gene
for glycosomal glyceraldehyde phosphate dehydrogensase
(gGAPDH) has been sequenced from over 100 trypanosomes to date [9]. This gene, though comparatively short
(800 bp), can be used for accurate phylogenetic placement
of novel trypanosomes as it is easy to align non-ambiguously without gaps. gGAPDH phylogenetic trees have
approximately the same resolution as those constructed
using complete (2000 bp) 18S rDNA sequences [9].
Figure 1. Schematic phylogenetic tree based on combined data from gGAPDH and
18S rRNA gene sequences [2,4,9]. The tree illustrates the phylogenetic
relationships and diversity of the tsetse-transmitted trypanosomes. Species or
genotypes discovered in the past 10 years are indicated with a red circle.
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Newly discovered diversity
When techniques of molecular identification have been
applied to field-collected samples, particularly those from
infected tsetse flies, previously unknown trypanosomes
have been found, and there are now at least ten new
trypanosomes that can be recognized (Table 1). The starting point for the expansion of diversity was the resolution
of two kinds of T. congolense, savannah and riverine-forest,
by isoenzyme analysis [10,11]. Subsequent development of
specific PCR tests and analysis of many field populations
has confirmed the association with the eponymous
habitats, and the riverine-forest type is typically found
in palpalis-group flies [10,12]. Two further types of T.
congolense were then described from Kenya: Kilifi (Kenya
Coast) [11,13,14] and Tsavo, later renamed T. simiae Tsavo
[5,15]). Despite significant genetic differences, none of
these new trypanosomes has been awarded species status,
and few studies have attempted to examine what phenotypic differences might exist.
The discovery of a previously unknown, tsetse-transmitted trypanosome in The Gambia, followed by its
thorough biological characterization, did prompt the designation of a new species, T. godfreyi [1]. The developmental
cycle of this trypanosome involved the midgut and proboscis, identifying it as a member of subgenus Nannomonas,
whereas experimental animal infection studies showed it
to be a suid trypanosome, like T. simiae, with which it
groups in phylogenetic analyses (Figure 1) [1]. Tsetse
surveys have revealed that T. godfreyi has a wide distribution and it has been found in both East and West Africa
[1,3,16].
Many of the new trypanosomes listed in Table 1 have
been found during field surveys of infected tsetse, and the
vertebrate hosts of some of these trypanosomes remain
unknown. For example, T. godfreyi-like was found during a
survey in Tanzania when trypanosomes that remained
unidentified after an initial screen using species-specific
PCR tests were followed up by DNA sequencing of the 18S
rRNA gene [3]. No live sample of this trypanosome was
collected for analysis, and its identity rests solely on molecular data.
With so many different kinds of trypanosome to identify,
species-specific PCR tests became too laborious, and have
been superseded by generic identification tools such as
FFLB. This has also solved the problem of the high
false-negative rate of species-specific PCR tests, which
sometimes failed to identify a greater proportion of trypanosome infections than they identified! These non-identified infections represent the hidden diversity of tsetsetransmitted trypanosomes, as demonstrated recently by
the discovery of a new T. brucei-like trypanosome in Tanzania, the Msubugwe trypanosome (Box 1) [2], and
increased diversity in the T. vivax group (Box 2) [4].
Prior to the introduction of generic identification tools,
many of the unidentified trypanosome infections might
reasonably have been expected to be reptilian trypanosomes [16,17] and therefore of no clinical importance.
Palpalis group flies often take a high proportion of blood
meals from large reptiles such as crocodiles and monitor
lizards, and these trypanosomes, for example T. grayi, the
crocodile parasite, have a stercorarian mode of develop-
Trends in Parasitology
Vol.xxx No.x
Box 1. The Msubugwe trypanosome
In 2006, a survey of trypanosomes carried by tsetse flies in coastal
Tanzania revealed the presence of a novel trypanosome [2].
Sequencing the genes for gGAPDH and 18S rRNA revealed that
this trypanosome was most closely related to T. brucei sensu lato
(Figure 1). The phylogenetic position shows that this trypanosome is
a distinct species to T. brucei s.l. because of the large genetic
distance, and might even represent a new subgenus [2]. The
trypanosome was present in 22% of midgut infections and 4.5% of
proboscis infections [4], making it one of the most common
trypanosomes in this area of Tanzania, with an equivalent
prevalence to livestock pathogens such as T. congolense savannah
or T. simiae. It is somewhat surprising that a trypanosome species
so closely related to a species of medical and veterinary importance
had not been previously recognized, especially since multiple
studies were conducted in this area [3,8,16]. However, speciesspecific PCR tests would have failed to identify this trypanosome,
and its presence in mixed infections would have gone unnoticed.
This trypanosome now provides an evolutionary link between
subgenus Trypanozoon and the other tsetse-transmitted trypanosomes. Its relatively close relationship to T. brucei makes it an ideal
candidate for comparative genomic studies.
The vertebrate hosts of the Msubugwe trypanosome and its
pathogenicity are unknown. Yet, it is possible that this trypanosome
is T. (Pycnomonas) suis (Table 2), a species first described in
Tanzania in sick pigs by Ochmann in 1905 [6], but then largely
forgotten. This trypanosome has a unique developmental cycle in
tsetse, colonizing the midgut, salivary glands and proboscis, where
it produces metacyclics [24].The position of the Msubugwe
trypanosome in the phylogenetic tree between subgenus Trypanozoon and Nannomonas is consistent with the position expected for
T. suis, as its developmental cycle in the tsetse fly combines features
of both (Table 2); however, there are no molecular data from T. suis
for comparison to the Msubugwe trypanosome.
A live specimen of the Msubugwe trypanosome was identified
among trypanosome isolates from midguts of wild-caught tsetse
flies (Emily R. Adams, Ph.D. thesis, University of Bristol, 2008) which
should help to answer some of the remaining biological questions.
Studies of trypanosome infections in livestock in the Tanga region
of Tanzania should elucidate further information regarding the host
range of this trypanosome.
ment where infective forms develop in the hindgut rather
than mouthparts. Indeed, T. grayi infections were identified from Glossina palpalis gambiensis in The Gambia [18]
and from G. fuscipes fuscipes in the Central African Republic [19] by hybridization with total DNA purified from
cultured T. grayi, although so far reptilian trypanosomes
have not been reported from studies using generic identification techniques.
Indeed, the problems of cryptic diversity and accurate
identification of species or genotypes are not confined to the
African trypanosomes. On the other side of the Atlantic, it
has taken several decades for the six major genetic lineages
of the human pathogen T. cruzi to be elucidated and
accepted, although they do not yet have taxonomic status
[20]. Likewise, unraveling the complex epidemiology of
species and subspecies within genus Leishmania also
relies heavily on genotyping by microsatellites, for
example [21]. Recent surveys have resulted in the discovery of high diversity of Plasmodium in wild chimpanzees,
which have provided important clues as to the origins of
human pathogenic species [22]. However, whereas the
majority of novel T. cruzi, Leishmania and Plasmodium
genotypes were isolated directly from vertebrate hosts or
human patients suffering from disease, the novel African
trypanosomes described here were mainly discovered
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Box 2. The Trypanosoma vivax complex
Trypanosoma vivax is one of the most economically important
pathogenic trypanosomes owing to the impact on livestock in
Africa, and also in South America, where it persists in a non-tsetse
transmission cycle. Differences in the pathogenicity of strains from
East and West Africa have long been recognized; strains in West
Africa tend to be more virulent, although haemorrhagic variants in
Kenya have been recognized which can cause catastrophic disease
[25]. Two species remained in subgenus Duttonella after the
taxonomic revisions of Hoare [6] (Table 2), but recent molecular
studies have revealed far higher levels of diversity. For example, an
initial species-specific PCR test developed for West African T. vivax
failed to recognize many T. vivax infections in livestock and tsetse in
East Africa and was quickly superseded by a PCR test targeted to a
conserved T. vivax gene [26]. Early molecular characterization
studies of relatively small numbers of isolates already hinted at
high levels of diversity [27,28], and this has been confirmed by
recent phylogenetic analysis gGAPDH genes showing at least three
clades present within subgenus Duttonella [4]. Two of these groups,
T. vivax A and B, were found by FFLB analysis of proboscis
infections of tsetse collected in Tanzania; both groups are phylogenetically distinct from the main cluster of T. vivax C isolates from
Africa and South America (Figure 1) [4]. Within T. vivax group C, the
South American isolates are surprisingly similar to those from West
Africa, and some isolates from West Africa are genetically homogeneous, as seen in a study of population structure of T. vivax from
The Gambia [29]; however, distinct isolates within this group have
recently been characterized from Kenya and Mozambique, and
pathogenicity of one of these isolates to goats has been demonstrated [30,31]. It is becoming clear that subgenus Duttonella shows
great diversity across its range in tropical Africa. Our limited
knowledge of the mammalian hosts, pathogenicity and response
to drug treatments of the various T. vivax genotypes hinders
attempts to design rational control programmes for trypanosomiasis caused by T. vivax.
circulating in the vector and thus are of unknown significance in terms of pathogenicity and epidemiology.
Does further diversity remain to be discovered within
the African trypanosomes?
It is clear from recent studies using generic methods such
as ITS-PCR and FFLB that the diversity of the African
trypanosomes is greater than previously thought. Do we
now expect to find further diversity?
Generic methods have only recently been applied to the
identification of trypanosome infections in field-collected
tsetse, but have already detected several new trypanosome
genotypes. The studies carried out thus far have been
limited in terms of tsetse species and geographical areas.
Considering that there are at least 30 species and subspecies of tsetse, many of which are restricted to tropical
forests, it is plausible that many more tsetse-transmitted
trypanosomes remain to be described. In particular, the flies
that inhabit dense forest, the fusca group, comprise more
than half of all described tsetse species [23]. As these flies
tend to feed on the wild mammals present in their forest
environment and seldom on livestock, they are generally
thought to pose little threat in terms of disease transmission
and hence are rarely investigated. Nonetheless, there may
well be unknown trypanosomes adapted to transmission
cycles involving these forest flies and wild mammals.
Encroachment of livestock into such natural transmission
cycles could promote the escape of these potential pathogens, allowing their wider dissemination in other tsetse
species with less restricted habitat requirements.
4
Trends in Parasitology Vol.xxx No.x
Could important pathogens remain undiscovered?
The discovery of the causative organisms of African trypanosomiases dates back over a hundred years. Among
this group of intensively studied trypanosomes, it seems
unlikely that important pathogens could have remained
undetected until now. Some of the novel trypanosomes
included here may represent the rediscovery of forgotten
species such as T. uniforme or T. suis, which are both
acknowledged pathogenic trypanosomes [6]. Generic
identification tools have been used mainly for tsetse
surveys to date, with few detailed studies on the trypanosomes present in livestock or wild animals. Most livestock
surveys have been carried out on cattle rather than small
ruminants, pigs or fowl, although these animals are ubiquitous and have important economic value for small
farmers in Africa. Indeed, pigs are a preferred food source
for tsetse, providing ample transmission opportunities for
the trypanosomes commonly found in tsetse, such as T.
godfreyi; however, there is as yet no natural host record for
this species, although it is presumably common in pigs
across the African continent.
Trypanosomiasis in African livestock is often characterized by very low parasitaemias and frequent mixed
infections, resulting in possible inaccurate diagnosis of
the causative organism(s). Reliable identification of
species relies on staining a thin blood smear rather than
observing live trypanosomes, and even then, many of the
trypanosomes in Table 1 are indistinguishable by
morphology. T. vivax A (Box 2) was the genotype most
commonly found in tsetse in Tanzania [4], making it
probable that this was the trypanosome encountered in
cattle diagnosed with T. vivax trypanosomiasis in Tanzania. Rare trypanosomes, those with a limited geographical
distribution or those with low parasitaemia might also
have escaped attention.
Thus there are several pertinent reasons why pathogenic tsetse-transmitted trypanosomes might have
been undiscovered to date. The more knowledge we have
concerning the full diversity of these trypanosomes, their
mammalian hosts and pathogenicity, then the better
chance we have of developing effective control measures.
Acknowledgements
Natural Environment Research Council and Royal Tropical Institute
(NERC).
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