genetic variation in taenia solium
TRANSCRIPT
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Parasitology International 5
Genetic variation in Taenia solium
Gillian Campbell a,*, Hector H. Garcia b, Minoru Nakao c, Akira Ito c, Philip S. Craig a
a Cestode Zoonoses Research Group, Bioscience Research Institute and School of Environment and Life Sciences, Salford University M5 4WT, UKb Department of Microbiology, Universidad Peruana Cayetano Heredia, Cysticercosis Unit, Instituto de Ciencias Neurologicas, Lima, Peru
c Department of Parasitology, Asahikawa Medical College, Midorigaoka-Higashi 2-1-1-1, Asahikawa 078-8510, Japan
Available online 13 December 2005
Abstract
Neurocysticercosis is a major zoonotic larval cestode infection that has a worldwide distribution and is of significant public health importance.
Knowledge of the genetic structure of Taenia solium can be applied to the epidemiology and transmission of this disease, since genetic variants
may differ in infectivity and pathogenicity. Molecular epidemiological approaches can also enable detailed studies of transmission. On a global
scale, mitochondrial markers have differentiated between T. solium isolates from Asia and isolates from Africa/Latin America. Intraspecific
variation in T. solium has been detected to some extent, using RAPD markers to differentiate between T. solium populations from different regions
within Mexico. Markers currently available for T. solium have not been used to analyse genetic variation at the community or local level. The
development of highly polymorphic markers such as microsatellites in T. solium can provide the means to examine genetic heterogeneity of
tapeworm infection at the household, community and regional level. Preliminary studies suggest it is possible to analyse population genetic
variation in communities using a range of polymorphic markers.
D 2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: Taenia solium; Cysticercosis; Molecular epidemiology; Polymorphism; Population genetics; Mitochondrial DNA; Microsatellite DNA
1. Introduction
The pork tapeworm Taenia solium is responsible for two
important diseases in humans, taeniasis and cysticercosis and is
the leading cause of non-congenital epilepsy in the developing
world. Humans are the only definitive host, and pigs are the
main intermediate host so there is great potential to eradicate
this disease [1]. T. solium has largely been eliminated from
Europe although it remains highly prevalent in endemic regions
of Asia, parts of Africa and Latin America. Indeed its
distribution may be spreading. In the USA T. solium may be
introduced with immigrants, refugees and tourists, as demon-
strated recently in New York [2], making this disease not only a
concern for developing countries.
Until recently genetic variation within the species T. solium
was a virtually unknown phenomenon, though there has been
increased interest in this area. DNA analysis of T. solium
isolates from different areas of the world indicates variation
between two broad geographic regions, Asia and Africa/Latin
1383-5769/$ - see front matter D 2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.parint.2005.11.019
* Corresponding author. Tel.: +44 161 2952094; fax: +44 161 295 5210.
E-mail address: [email protected] (G. Campbell).
America [3]. More is known about its inter-specific variation,
which has been used to differentiate T. solium from Taenia
saginata and Taenia asiatica. DNA analysis has proven to
have more discriminatory power than morphological character-
istics [3].
Knowledge of the genetic structure of T. solium can be
applied to the epidemiology and transmission of these
parasites, since genetic variants may differ in their infectivity
and pathogenicity. Furthermore, analysing genetic variation
within and between populations can provide information
about future evolutionary change, genetic differentiation and
speciation [4]. As tapeworms are hermaphrodites capable of
self-fertilising, their progeny are likely to be genetically
identical, i.e. clones. This characteristic has a great impact on
the genetic structuring of tapeworm populations. For example,
self-fertilisation may depress genetic variation within popula-
tions, and increase differentiation among populations [5].
Ability to differentiate clonal populations of T. solium would
provide important information on the epidemiology at
household, village, local and regional levels. For example,
the identification of larval populations in pigs derived from
the same Taenia carrier within the community is useful to
evaluate transmission patterns.
5 (2006) S121 – S126
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G. Campbell et al. / Parasitology International 55 (2006) S121–S126S122
2. Molecular tools
The advance of molecular techniques has helped develop-
ment of new tools and approaches in the field of parasitology,
including the taeniid tapeworms Taenia and Echincococcus
[3,6]. Molecular approaches have greatly impacted the identi-
fication and systematics of parasites, the diagnosis of infections,
epidemiology and transmission patterns, analysis of population
subdivision and the study of drug resistance and vaccine
development [7]. The first notable application of genetic
markers in parasitology was the investigation of genetic
variation in the fluke Fascioloides magna [8] and in the
nematode Ascaris [9]. Mitochondrial DNA sequence variation
revealed 42 genotypes in 62 Ascaris worms from 35 households
in 3 Guatemalan locations. Significant heterogeneity was found
among populations from different villages, but not between
families from the same village. Studies in Echinococcus spp.
have enabled discrimination between strains/genotypes (10
currently recognised for Echinococcus granulosus), and eluci-
dation of transmission cycles [10,11].
There are now many widely accessible DNA and PCR
based tools available to parasitologists and many of them have
been discussed and reviewed in detail [7,12,13]. Some of the
most commonly used markers that are referred to later in this
paper are summarised below.
2.1. Mitochondrial DNA
Mitochondrial DNA (mtDNA) sequences are frequently used
for the study of phylogenetics and population differentiation.
MtDNA sequences evolve at a faster rate than nuclear genes and
are therefore more suited for discriminating closely related or-
ganisms [14].MtDNA is considered to be the best neutral marker,
having high levels of variability that can be easily quantified
without selection pressure from the host. Cytochrome c oxidase 1
(cox1) and NADH dehydrogenase subunit 1 (nad1) genes have
been sequenced in Taenia spp. and shown that Asian Taenia
could be considered a distinct species [15,16]. Furthermore,
phylogenetic analysis using cox1 sequences have resolved the
relationship between T. solium, T. saginata and T. asiatica [17].
Cytochrome b (cob) gene has also differentiated between global
genotypes of T. solium and between T. saginata and T. asiatica
[3,18]. Moreover, the polymorphism of Echinococcus mtDNA
suggests that the genotypes G4 (horse strain) and G5 (cattle
strain) of E. granulosus should be given species status [11,19].
2.2. Nuclear DNA
The most commonly used nuclear DNA fragment is the
internal transcribed spacer (ITS rDNA) region, comprised of
ITS1 and ITS2, separated by 5.8S and flanked by 18S and 28S
rRNA genes. ITS is a multigene family with tandemly arrayed
sequence repeats and been successfully used to detect strain
variation in Echinococcus [20]. Direct sequence analysis from
nuclear genes may not be sufficiently variable. Instead comple-
mentary tools are used in addition such as PCR-RFLP, SCCP,
RAPD and microsatellite analysis (see below for details).
2.3. PCR-RFLP
Restriction fragment length polymorphism (RFLP) detects
genetic polymorphisms in amplification products from a
chosen gene that have been digested by restriction enzymes
with specific recognition sequences. If mutations in the
sequence alter these recognition sites then the resulting
digestion profile will not detect a fragment for that site.
PCR-RFLP of the ITS rDNA region has been effectively used
to discriminate between Taenia spp. [21] and to help define
strains of Echinococcus [22].
2.4. SSCP
Single-strand conformation polymorphism (SSCP) relies on
the principle that the electrophoretic mobility of single-
stranded DNA is dependent on its structure and size [23].
Single-stranded molecules take on secondary or tertiary
structures (conformations) due to self-annealing between
nucleotides within each strand. The length, location and
number of these conformations vary and are used as a measure
of differentiation. PCR-based SSCP using an appropriate target
gene demonstrated intra-specific variation amongst some
Taenia spp. using nad1 and cox1 and had potential for
studying genetic structure of taeniid cestode populations [24].
2.5. Whole genome
2.5.1. RAPD
In the analysis of random amplified polymorphic DNA
(RAPD), short primers of arbitrary sequence are used to
amplify random anonymous genomic sequences [25]. These
primers are typically 10 bases long and are available in
commercial kits. RAPD analysis detects size variation among
the fragments but is insensitive to sequence differences.
Variation is characterised simply as the presence or absence
of a fragment. RAPD has been successfully employed for a
wide range of protozoan and metazoan parasites. Some genetic
diversity has been demonstrated in T. solium using RAPD
markers [26,27].
2.5.2. Microsatellites
Avaluable and now commonly applied tool for investigating
population structure is microsatellites [28,29]. Microsatellites
consist of short tandemly repeated DNA motifs that are widely
distributed throughout most of the eukaryotic genome [30]. The
DNA repeat motif (between 2 and 5 bases) varies in number at a
given locus, often hypervariable, generated by high mutation
rates. Furthermore, high variation at microsatellite loci has been
described in species in which little genetic diversity has been
detected by other less polymorphic genetic markers, for example
Echinococcus multilocularis [31].
3. DNA polymorphism in the genus Taenia
Until recently there has been very little research into genetic
variation within T. solium , compared to the numerous
G. Campbell et al. / Parasitology International 55 (2006) S121–S126 S123
investigations into E. granulosus, responsible for cystic
echinococcosis (e.g. [6,32–34]). Studies that have been done
indicate that there is little intra-specific variation in T. solium,
although it may not be the case for other taeniids. There is a
similar contrast observed between E. granulosus, for which 9–
10 different strains/genotypes have been identified, and the
relatively limited variability of E. multilocularis. The existence
of sufficient interspecific variation between T. solium, T.
saginata and T. asiatica has been used to differentiate between
these medically and economically important taxa. Indeed
phylogenetic analysis of mtDNA was able to distinguish
between T. saginata and T. asiatica and place them in a
separate clade to T. solium [17].
3.1. Intraspecific variation in T. solium
MtDNA markers have demonstrated little or no genetic
variability amongst T. solium. Evidence from cox1, cob and
nad1 sequences suggests that there is limited variability
amongst isolates from different countries. Southern blot
hybridisation demonstrated the first intra-specific sequence
variation between cysts of T. solium from India, Zimbabwe and
Mexico, although variability was not detected among the 6
isolates from India [35]. The short sequences of cox1 and nad1
revealed that the variation amongst T. solium isolates was
significantly less than the variation observed between species
[16,36]. However, variation was detected within 5 isolates of
Taenia taeniaeformis [36] (Table 1).
Analysis of the nad1 gene detected greater interspecific
variation for Taenia spp. than for the cox1 gene, and this gene
may have potential for determining population genetic structure
in the family Taeniidae [16]. Intraspecific variation was detected
amongst 7 Polish isolates of Taenia hydatigena using the nad1
gene [37]. In contrast, the cox1 gene was identical for 15 isolates
from Peru, even between regions as diverse as the jungle in
northeast and the southern highlands [38]. Moreover, cox1
sequences demonstrated little variation between Latin American
isolates of T. solium fromMexico, Columbia and Peru with only
Table 1
Summary table of the genetic variation detected within Taenia solium to date,
showing the number of isolates, the molecular marker, the method used,
polymorphism at the international and national level, and the reference
No. of
isolates
Molecular marker Method Polymorphisma Reference
International National
cDNA Hybridisation Yes No [35]
Cox1 Sequence Yes N/A [15]
Cox1 Sequence Yes N/A [36]
ND1 and Cox1 Sequence Yes N/A [16]
ND1 and Cox2 SSCP Yes N/A [24]
18 ITS 1 and 2 PCR-RFLP No No [39]
15 Cox1, ITS and Ts14 Sequence No No [38]
Cox1 and Cytb Sequence Yes No [18]
ITS 1 and 2 PCR-RFLP No N/A [40]
13 Cox1 and Cytb Sequence Yes No [3]
160 RAPD PCR Yes Yes [26]
90 RAPD PCR Yes Yes [27]
a Polymorphism measured between isolates between countries and within the
same country.
1 to 4 nucleotide changes differentiating them, although isolates
from India, China and the Philippines were unique [18].
In contrast, PCR-RFLP analysis of the complete ITS region
revealed no intraspecific variability within T. solium isolates,
despite this gene usually detecting intraspecific variation
amongst many species [39,40]. Intraspecific variation was
however detected amongst T. saginata isolates [40].
3.2. Global variation in T. solium
Recent phylogenetic analysis of complete sequences of cox1
and cob genes from 13 worldwide T. solium isolates suggests
that there are 2 distinct genotypes in T. solium, which
correspond to either an Asian or Latin American/African origin
isolate [3,18]. There have been conflicting theories regarding
the origin of T. saginata and T. solium and when humans
acquired this tapeworm. Polymorphism that is restricted to the
geographic origin of T. solium provided evidence to support the
theory that Taenia in humans predates the development of
agriculture, animal husbandry including the domestication of
pigs and cattle [41,42]. This contradicts previous suggestions
that humans acquired Taenia with the domestication of cattle
and pigs alongside the coincidental dissemination and coloni-
sation of humans, approximately 10,000 years ago (e.g.
[43,44]). Divergence of these 2 genotypes was estimated
following Despres and others [45] at 0.4–0.8 MYA for the
cox1 gene and 0.8–1.3 MYA for the cob gene, using the
divergence rate between mouse and rat of 9–12 MYA [46].
This would indicate that intraspecific variation occurred in T.
solium before the domestication of cattle and pigs [3].
The different origins for T. solium from China or Latin
America/Africa differentiate this species into the 2 genotypes
observed. However, variability amongst African and Latin
American isolates is lower than it is amongst isolates from
China [2,18]. Both cox1 and cob have identical sequences
between several paired locations in Africa and Latin America,
despite not being geographically close, suggesting that the
isolates were introduced recently. However, to date isolates
from China show rather limited sequence variation [18].
Sequence divergence amongst Asian isolates (0.1–0.8%)
reflects minor changes that occurred in different regions in
Asia, whereas low sequence divergence amongst Latin Amer-
ican isolates (0–0.6%) may be caused by introductions from
different regions of Europe [3]. This supports the speculation
that T. solium was introduced into Latin America and Africa
from Europe during the colonial period about 500 years ago and
that T. solium of another origin spread independently in Asia
[3]. The Asian T. solium genotype may have emerged in the
southern region of China where domestication of pigs started
independently from Europe [47].
The broad genetic differentiation in T. solium correlates well
with the clinical variation of patients with cysticercosis. Disease
manifestations due to neurocysticercosis (NCC) vary depending
upon the location, growth, size, number of cysts, and the
viability of lesions, stage of cyst degeneration and presence of
calcifications [48]. Cysticercosis in the muscle, subcutaneous,
eye and other tissues/organs, is referred to as extraneuronal or
G. Campbell et al. / Parasitology International 55 (2006) S121–S126S124
subcutaneous cysticercosis (SCC). In Latin America patients are
usually found predominantly with NCC, whereas in Asia
subcutaneaous cysticercosis is quite common and NCC usually
coincides with SCC. In Africa however the situation is far more
complicated with SSC rare in some regions, while in others
NCC and SCC occur as common as in Asia [47].
3.3. Population genetic analysis of T. solium
The population genetic structure of T. solium was examined
in Madagascar and Mexico [27]. Using RAPD markers, 49 out
of 113 loci (43%) were polymorphic between T. solium cysts
from the two countries. Nei and Li’s genetic distance was
measured at 0.2 [27]. The genetic distance dropped to 0.05
between central (CE) and southeastern (SE) Mexico, and
among 3 regions within CE Mexico genetic distance ranged
from 0 to only 0.01. Population genetic analysis showed low
genetic differentiation among the 3 localities in CE Mexico,
whereas there was higher genetic differentiation between
Mexico and Madagascar. Genetic differentiation was also high
both within and among individuals from CE and SE Mexico
due to the great geographic distance, resulting in little contact
between T. solium from these regions. Low gene flow can be
attributed to the low levels of migration of human carriers and
pig commerce between countries and between regions in
Mexico [27]. These authors also suggested this significant
variation between Mexico and Madagascar may effect host
preference and explain the differences in prevalence of NCC
and SCC between the two countries.
A very similar study investigated genetic variability in T.
solium from 3 regions in Mexico, Honduras and Tanzania [26].
Out of 49 RAPD loci, 88% were polymorphic in the 160 cysts
examined from 6 pigs. Variation was detected among and
within isolates with several fixed alleles in each, likely to have
been caused by genetic drift and inbreeding effects in the
hermaphrodite worm while in the definitive host. The
observation of genetic variability between 2 isolates from the
same community in Morelos, Mexico [26] is of interest to those
looking for variation at the community level.
So far, population genetic data from RAPD and the ITS
genes proves that genetic variation does indeed exist in T.
solium, suggesting it may have a role in variation in disease
presentation due to self- or cross-fertilisation [26]. This genetic
variability has important epidemiological implications, such as
the development of the parasite, its pathogenicity and drug
susceptibility. For instance, variation in populations of E.
granulosus has contributed to the identification of different
strains and helped elucidate transmission patterns.
3.4. Comparative studies in E. multilocularis
As with T. solium, little variation has been detected within
the worldwide population of E. multilocularis with the genetic
markers so far used. ITS1 and ITS2 sequences between 9
isolates from 5 German states were identical and even between
Germany, Japan and Alaska there was only one polymorphic
site [49]. The authors claimed this high degree of genetic
conservation in ITS1 and ITS2 contrasted with previous studies
where greater variability had been detected in other Echino-
coccus species. Development of more sensitive molecular tools
such as microsatellites and single nucleotide polymorphisms
(SNPs) that can detect genetic variation in small populations
may be required [50]. Recently, 2 microsatellites of trinucle-
otide motif FCAC_ type were developed in E. multilocularis,
detecting a total of 6 alleles that differentiated between
heterozygotes and homozygotes [31]. This paper is of interest
to those seeking to measure genetic variation in T. solium. It
shows that microsatellites are useful for population-level
polymorphisms even within a limited region where previously
mtDNA markers demonstrated limited variability [3]. Further-
more, a microsatellite fragment was isolated in E. multi-
locularis that detected 11 genotypes and was sensitive enough
to differentiate between Alaskan and European E. multi-
locularis isolates and also amongst European isolate clusters
[51]. From this new evidence in E. multilocularis, it is hopeful
that other, more sensitive markers can detect polymorphisms in
T. solium at a local population level.
4. Future prospects
Preliminary work has identified several molecular markers
from mtDNA for SNPs and PCR-RFLP, and microsatellites in
T. solium (Campbell et al., unpublished). These markers will be
utilised in analysing population genetic variation in T. solium
isolates from communities in Peru, Mexico and China. DNA
polymorphism has been detected between a panel of Peruvian
T. solium isolates using the three markers: 1) PCR-RFLP of the
gene region cob detected 4 alleles amongst the isolates; 2) One
optimised FCAC_ repeat loci detected 2 genotypes amongst the
isolates tested. A further 9 microsatellite loci have yet to be
tested; 3) SNP’s from atp6, cox2, cox3, and nad2 sequences
detected at least 4 genotypes amongst isolates.
Molecular techniques such as these can provide the much-
needed epidemiological data regarding the transmission of
taeniasis/cysticercosis in Asia, Africa and Latin America.
Examining genetic heterogeneity of tapeworm infection at the
family/household, community and eventually at geographical/
regional levels will help determine human exposure and
transmission patterns in endemic regions. More specifically it
may help address questions such as whether there is a single
tapeworm clone or several clones transmitted within a commu-
nity, where they originate, if the same clones reappear post-
treatment, and whether there are any spatial patterns to clone
presence regarding household and/or pig clusters.
Acknowledgements
Collection of samples from Peru was part of a collaborative
grant from the Burroughs Wellcome. Molecular analysis of
Taenia solium was also undertaken at Asahikawa Medical
College, Japan, funded in part by a Royal Society Japan–UK
Joint Project Grant (ref: 16362) and by a Grant-in-Aid for
International Collaboration Research from the Japan Society
for Promotion of Science (14256001, 17256002) to A. Ito.
G. Campbell et al. / Parasitology International 55 (2006) S121–S126 S125
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