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Ai et al. Parasites & Vectors 2011, 4:101
http://www.parasitesandvectors.com/content/4/1/101
REVIEW
Open Access
Genetic characterization, species differentiation
and detection of Fasciola spp. by molecular
approaches
Lin Ai1,2,3†, Mu-Xin Chen1,2†, Samer Alasaad4, Hany M Elsheikha5, Juan Li3, Hai-Long Li3, Rui-Qing Lin3,
Feng-Cai Zou6, Xing-Quan Zhu1,6,7* and Jia-Xu Chen2*
Abstract
Liver flukes belonging to the genus Fasciola are among the causes of foodborne diseases of parasitic etiology.
These parasites cause significant public health problems and substantial economic losses to the livestock industry.
Therefore, it is important to definitively characterize the Fasciola species. Current phenotypic techniques fail to
reflect the full extent of the diversity of Fasciola spp. In this respect, the use of molecular techniques to identify
and differentiate Fasciola spp. offer considerable advantages. The advent of a variety of molecular genetic
techniques also provides a powerful method to elucidate many aspects of Fasciola biology, epidemiology, and
genetics. However, the discriminatory power of these molecular methods varies, as does the speed and ease of
performance and cost. There is a need for the development of new methods to identify the mechanisms
underpinning the origin and maintenance of genetic variation within and among Fasciola populations. The
increasing application of the current and new methods will yield a much improved understanding of Fasciola
epidemiology and evolution as well as more effective means of parasite control. Herein, we provide an overview of
the molecular techniques that are being used for the genetic characterization, detection and genotyping of
Fasciola spp..
Background
Fascioliasis is an important food-and water-borne parasitic zoonosis caused by liver flukes of the genus Fasciola
(Platyhelminthes: Digenea: Fasciolidae) [1,2]. Fasciola
spp. have a cosmopolitan distribution, with high frequency in tropical areas [1,3,4]. Human fascioliasis has
been reported in numerous countries [1,3,5]. It is estimated that millions of people are infected worldwide and
the number of people at risk exceeds 180 million [6].
Also, fascioliasis is one of the most important parasitic
diseases in grazing animals with over 700 million production animals being at risk of infection and economic
* Correspondence: xingquanzhu1@hotmail.com; chenjiaxu1962@163.com
† Contributed equally
1
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of
Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research
Institute, CAAS, Lanzhou, Gansu Province 730046, P R China
2
National Institute of Parasitic Diseases, Chinese Center for Disease Control
and Prevention, Shanghai 200025, P R China
Full list of author information is available at the end of the article
losses were estimated at > US$ 2 billion per year worldwide [6].
A few species have been described within the genus
Fasciola, but only three species, Fasciola hepatica, Fasciola gigantica and Fasciola jacksoni are commonly
recognized as taxonomically valid, with F. hepatica
mainly occurring in temperate areas, F. gigantica in tropical zones, and both taxa overlapping in subtropical areas
[6-11]. F. jacksoni is known as the fasciolid of Asian elephants and its phylogenetic position is still uncertain
[12]. Given the adverse impact of Fasciola infection on
human health and its economic significance, rapid and
accurate identification of Fasciola species is necessary for
successful clinical management of infection, and for epidemiological surveys.
For a long time, the identification of Fasciola spp. has
been based solely on traditional morphological approaches.
However, due to the limitations of morphological methods,
various molecular approaches have been developed and
used for the identification and differentiation of Fasciola
© 2011 Ai et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Ai et al. Parasites & Vectors 2011, 4:101
http://www.parasitesandvectors.com/content/4/1/101
Page 2 of 6
species. Importantly, these molecular methods have raised
questions and spurred debate on the recognition of the
“intermediate Fasciola“ as a hybrid/introgressed form
between F. hepatica and F. gigantica [8,13-20]. This hybrid
Fasciola represents the emergence of a natural diversity
previously undetected using conventional approaches,
probably because of the inadequacy of their discriminatory
power.
This article reviews molecular techniques used to
identify and detect genetic variation among Fasciola spp.
The identification and recognition of the “intermediate
Fasciola“
A number of genetic and phylogenetic studies (Table 1)
using different molecular targets have shown the existence of novel “intermediate Fasciola“ [13-19,21-23].
Ribosomal DNA (rDNA) is one of the most useful
markers in genetic studies because it is available in high
copy number and contains variable regions flanked by
more conserved regions [24]. Previous studies have
demonstrated that the first and second internal transcribed spacers (ITS-1 and ITS-2) of rDNA located
between the nuclear small and large subunit rRNA genes
can provide genetic markers for species-level identification of Fasciola [14,18,21,22,25] (Table 1). The ITS-2
sequence motifs were considered the DNA barcodes for
Fasciola spp. [26]. Comparing ITS-2 sequences, six sites
at which F. gigantica and F. hepatica differ were found,
and one of these is a deletion in F. gigantica relative to F.
hepatica [18,22,25,27]. Whereas, the “intermediate Fasciola“ has nucleotides shared between the two Fasciola
species. In agreement with results obtained by using ITS2 sequences, F. gigantica was found to be different from
F. hepatica at five nucleotide positions in the ITS-1
sequences, whereas the “intermediate Fasciola“ has the
ITS-1 sequence of both F. gigantica and F. hepatica
[15-17,21]. In addition to the ITS sequences, the D2
region of 28S rDNA provided genetic evidence for the
existence of natural hybridization between F. gigantica
and F. hepatica in Korea [18].
Table 1 Summary of molecular approaches used for the detection and/or genetic differentiation of Fasciola spp..
Molecular
approach
Species investigated
F. hepatica
Developmental stage
DNA target regions
References
Intermediate
form
Adult
√
ITS2
38
√
√
√
√
ITS2
14, 27
√
√
√
ITS1, ITS2
25
√
√
√
√
√
√
nad1, cox1
nad1, cox1
28
13, 17
√
√
√
√
ITS2, cox1
9
√
√
√
√
ITS1, nad1
16
√
√
√
√
cox1, ITS1, ITS2
15
√
Conventional PCR
F. gigantica
√
√
√
nad1, cox1, ITS2, 28S
18
√
Complete mitochondrial genome
30
√
124 bp repetitive DNA sequence
49
√
124 bp repetitive DNA sequence
28S
50
39
cox1, ITS1, ITS2
52
ITS2
47
√
√
√
√
√
Cercaria
Eggs
√
Multiplex PCR
√
Specific PCR
√
√
√
√
√
PCR-RFLP
√
√
√
√
ITS2
22
√
cox1, ITS2
23
√
ITS1
8, 21
√
√
√
√
√
√
√
Repetitive DNA sequences
46
√
PCR-SSCP
√
√
√
√
√
Random nucleotide sequence
Random nucleotide sequence
43
53
√
Random nucleotide sequence
42
√
√
√
Random nucleotide sequence
10
Repetitive DNA fragments
48
ITS2
54
RAPD-PCR
SRAP
√
√
√
√
√
DNA probe
√
TaqMan real-time PCR
√
√
√
√
LAMP
√
√
√
√
PCR
√
√
√
√
IGS
58
Microsatellites
59
Ai et al. Parasites & Vectors 2011, 4:101
http://www.parasitesandvectors.com/content/4/1/101
Other molecular markers being used frequently are
mitochondrial DNA (mtDNA) sequences. Almost all of
eukaryotes contain a mitochondrial genome which
evolves at a faster rate than the nuclear genome and, is
thus suitable for discriminating closely related organisms
[28-30], especially at the species and sub-species levels
[31-33]. mtDNA sequence analysis also provided evidence for the existence of the “intermediate Fasciola”
[15,16,19].
Genetic variation among and within Fasciola spp
Revealing genetic variation has been the focus of many
studies because accurate analysis of genetic variability
has important implications for studying population biology, epidemiology, and genetic structure of these parasites, and thus the efficient control of the diseases they
cause.
Examination of Fasciola specimens from geographical
areas where F. hepatica and F. gigantica co-exist, such
as Egypt and Iran, demonstrated the existence of phenotypic variations in adult flukes [20,34]. Molecular
approaches utilizing a number of genetic makers are
useful for genetic characterization and studies of genetic
variability among parasite populations [35-37]. A recent
study investigated the extent of genetic variation among
Fasciola collected from different host species and geographical localities in Spain using ITS rDNA as genetic
markers, and concluded that only a single species
F. hepatica exists in Spain, although a slight sequence
variation in the ITS-2 was detected among F. hepatica
samples from different host species and geographical
areas [38]. Spanish F. hepatica examined in that study
differed from F. hepatica from elsewhere by two nucleotides in the ITS-2 [38]. Another study detected some
genetic variations in F. hepatica from northwest of
Spain using the 28S rDNA as genetic marker, and there
were nucleotide differences in a number of sequence
positions [39].
Sequence related amplified polymorphism (SRAP) is a
molecular technique for detecting genetic variation in
the open reading frames (ORFs) of genomes of related
organisms [40,41]. A recent study showed that the
SRAP technique was useful for revealing genetic variability within and between F. hepatica, F. gigantica and
the “intermediate Fasciola”, which substantiated the evidence for the existence of the “intermediate Fasciola”
[10]. Using the same technique, genetic variability
among a number of F. hepatica samples collected from
six host species and 16 geographical locations in Spain
was investigated [42], and a low genetic variation in the
coding regions of the genomes was found, indicating the
lack of genetic association between F. hepatica and their
hosts and/or geographical locations in Spain [42].
Page 3 of 6
Randomly amplified polymorphic DNA (RAPD) is a
useful genetic marker for the identification and genetic
characterization of parasite populations [37]. RAPD is a
useful technique for the identification and differentiation
of F. hepatica and F. gigantica. Using the RAPD technique, some degree of genetic variation was detected
among F. gigantica isolates from cattle, buffalo, and
goat. Cattle and buffalo isolates of F. gigantica showed
100% homogeneity, whereas goat and cattle/buffalo isolates displayed 92.68% similarity [43].
mtDNA sequences provided useful markers for studies
of genetic variability and population structures [28,44].
Walker et al. (2007) examined DNA polymorphism in
the entire mitochondrial genome and showed that
genetic diversity exist among and within F. hepatica
populations from cattle and sheep and provided evidence for the existence of multiple mitochondrial
lineages within infra-populations of F. hepatica [44].
Another study examined genetic variation in eastern
European and western Asian populations of F. hepatica
using partial mitochondrial NADH dehydrogenase subunits 1 (nad1) and cytochrome c oxidase subunit 1 gene
(cox1) as genetic markers, and revealed the existence of
two well-defined lineages with two main haplotypes and
a number of shared divergent haplotypes among the
examined F. hepatica populations [28].
Molecular detection and identification of Fasciola spp
In order to overcome the limitation of the phenotypic
methods, genotypic approaches have been used for the
identification and differentiation of Fasciola spp.
[9,10,21,22,45-47]. Before the availability of PCR-based
approaches, DNA probes were the alternative choice for
the genotypic detection of Fasciola spp. [48]. However,
DNA probe-based assays usually require the use of radioactive isotopes and can have bio-safety concerns.
Over the last two decades, several PCR-based
approaches (Table 1), including PCR-linked restriction
fragment length polymorphism (PCR-RFLP), PCR-linked
single-strand conformation polymorphism (PCR-SSCP)
and specific PCR assays, have been developed for the accurate identification of Fasciola spp. [8,21,22,45,46]. For
example, a simple and rapid PCR-RFLP assay targeting a
618-bp sequence of the 28S rDNA was developed for the
differentiation between F. hepatica and F. gigantica [45]. A
similar PCR-RFLP assay using restriction endonucleases
Hsp92 II and Rca I was developed to differentiate between
F. hepatica, F. gigantica and the “intermediate Fasciola” in
China utilizing the ITS-2 rDNA as genetic marker [22].
Using the ITS-1 rDNA as genetic marker, Lin et al. (2007)
established a PCR-SSCP assay for the accurate identification and differentiation between F. hepatica, F. gigantica
and the “intermediate Fasciola” [21]. A very recent study
Ai et al. Parasites & Vectors 2011, 4:101
http://www.parasitesandvectors.com/content/4/1/101
established a fluorescence-based SSCP (F-PCR-SSCP) for
the identification of Fasciola spp. [46].
Recently, several specific PCR assays have been developed to differentiate F. hepatica from F. gigantica and
detect Fasciola infections in the intermediate host snail
and definitive hosts (such as buffalo), utilizing various
genetic markers, such as cox1, ITS, non-coding repetitive DNA fragment as well as RAPD-derived sequences
[47,49-53]. For example, Ai et al. (2010) established a
specific PCR method based on the ITS-2 sequences to
identify F. hepatica, F. gigantica and the “intermediate
Fasciola”. The method was sensitive as it was able to
amplify target DNA fragment from a single Fasciola egg
[47]. Specific PCR assays, using two primer sets derived
from RAPD-derived sequences from English F. hepatica
and Ghanaian F. gigantica, were able to distinguish
F. hepatica from F. gigantica from cattle and sheep
hosts from different countries [53]. Alasaad et al. (2011)
developed a highly specific, sensitive, and simple TaqMan-based real-time PCR assay for the identification of
F. hepatica and F. gigantica, as well as the “intermediate
Fasciola” based on sequences of the ITS-2 rDNA [54].
A PCR assay was used to detect F. gigantica infection
in the snail vector host, Lymnaea auricularia [49]. The
specific primers amplified a F. gigantica specific 124-bp
non-coding repetitive DNA fragment from infected
L. auricularia snails. Kaplan et al. (1995) also identified
a 124-bp repetitive DNA sequence which was used as a
specific probe for detection of F. hepatica infections in
intermediate host snails (Fossaria cubensis and Pseudosuccinea columella) [51]. A multiplex PCR assay was
able to detect F. hepatica DNA in L. viatrix snails,
which were even formalin-fixed and paraffin-embedded
[52]. TaqMan chemistry was adopted by Schweizer et al.
(2007) to establish a real-time PCR assay. The combined
use of primers and probe targeting an 86-bp target of a
repetitive 449-bp genomic DNA fragment facilitated
the detection of L. truncatula naturally infected with
F. hepatica. These PCR assays are highly specific and
sensitive, providing useful and practical tools for the epidemiological investigation of Fasciola in the snail hosts
[55].
Loop-mediated isothermal amplification (LAMP)
allows amplification of target nucleic acids under isothermal conditions with high sensitivity, specificity,
rapidity and precision, which has found broad applications for the detection of pathogens [56,57]. Ai et al.
(2010) developed a LAMP assay for the sensitive and
rapid detection and discrimination of F. hepatica and
F. gigantica. The assay can be done in 45 min under isothermal conditions at 61°C or 62°C by employing a set
of 4 species-specific primer mixtures and the results can
be checked visually. This LAMP assay was approximately 105 times more sensitive than the conventional
Page 4 of 6
specific PCR assays, and may find applicability in the
field settings or in poorly-equipped laboratories in endemic countries [58].
Conclusions and future perspectives
Much of the current knowledge of Fasciola spp. taxonomy and epidemiology has stemmed from numerous
observational and morphological studies. However, conventional methods of detection and differentiation of
Fasciola do not accurately reflect the full diversity of
Fasciola spp. Nevertheless, molecular genetics studies
over the past two decades have added significantly to
our understanding of Fasciola taxonomy, genetics, and
contributed to the development of advanced approaches
for the accurate identification and differentiation of Fasciola spp. Importantly, these molecular methods have
facilitated the identification of the hybrid “intermediate
Fasciola”. However, presently there is no molecular
diagnostic method (e.g. a Copro-PCR) developed and
validated for use with human stools.
We are still far from a complete understanding of the
molecular evolution of the hybrid Fasciola, and many
questions remain unanswered. For example, what is the
outcome of experimental crosses between F. hepatica
and F. gigantica? What are the differences between the
“intermediate Fasciola” and other Fasciola species at the
genomic and transcriptomic levels? Comprehensive
genetic characterization using more variable markers
such as microsatellites (eg. [59]) along with transcriptional analysis of Fasciola species can be used to refine
the taxonomic status of the “intermediate Fasciola” and
to assess its potential as a zoonotic agent.
On the analytical methods front, there is a clear need
for the application of high-throughput molecular techniques such as next-generation sequencing, transcriptomics, proteomics and large-scale analysis of single
nucleotide polymorphisms. The successful application of
these and other techniques should bring more insights
into the population genetic structure and the evolutionary process in Fasciola.
Recently, there have been increasing interests in the
studies of transcriptome and proteome of F. hepatica
[60-62]. More studies in these promising areas of
research will expand our understanding of the complex
biology of different Fasciola species, which, in turn, will
facilitate the development of novel means of therapeutic
and immunological intervention.
Acknowledgements
This work was supported, in part, by the National S & T Major Program
(Grant No. 2008ZX10004-011, 2009ZX10004-302, 2009ZX10004-201), the
National Key Technology R & D Program (Grant No. 2008BAI56B03), the State
Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research
Institute, Chinese Academy of Agricultural Sciences, the Yunnan Provincial
Program for Introducing High-level Scientists (Grant No. 2009CI125) and the
Ai et al. Parasites & Vectors 2011, 4:101
http://www.parasitesandvectors.com/content/4/1/101
Page 5 of 6
Program for Changjiang Scholars and Innovative Research Team in University
(Grant No. IRT0723).
13.
Author details
1
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of
Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research
Institute, CAAS, Lanzhou, Gansu Province 730046, P R China. 2National
Institute of Parasitic Diseases, Chinese Center for Disease Control and
Prevention, Shanghai 200025, P R China. 3College of Veterinary Medicine,
South China Agricultural University, Guangzhou, Guangdong Province
510642, P R China. 4Estación Biológica de Doñana, Consejo Superior de
Investigaciones Científicas (CSIC), Avda. Américo Vespucio s/n 41092 Sevilla,
Spain. 5School of Veterinary Medicine and Science, University of Nottingham,
Sutton Bonington Campus, Loughborough, LE12 5RD, UK. 6College of
Animal Science and Technology, Yunnan Agricultural University, Kunming,
Yunnan Province 650201, P R China. 7College of Animal Science and
Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing,
Heilongjiang Province 163319, P R China.
14.
15.
16.
17.
Authors’ contributions
XQZ, JXC, SA and HME conceived and designed the review, and critically
revised the manuscript. LA and MXC drafted the manuscript. JL, HLL, RQL
and FCZ contributed to drafting the manuscript. All authors read and
approved the final manuscript.
18.
Competing interests
The authors declare that they have no competing interests.
19.
Received: 19 March 2011 Accepted: 10 June 2011
Published: 10 June 2011
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Cite this article as: Ai et al.: Genetic characterization, species
differentiation and detection of Fasciola spp. by molecular approaches.
Parasites & Vectors 2011 4:101.
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