molecular approaches to study Fasciola species

1. Molecular Approaches for Studying
Fasciola Species
Course No: VPAR-609 2T
Course Title: Molecular and Biochemical Parasitology
MD. AKHTARUZZAMAN
ID No.: 17VPARAJJ12M
Reg. No.: 38443; Session: 2011-12

2. Introduction
Molecular methods are ideal for species
identification, strain confirmation and population
genetic studies. Most of these molecular methods
are polymerase chain reaction (PCR) dependent
targeting a common gene which is adequately
divergent between taxa.
The targeted region must be long enough to provide
adequate variable characters to appreciate
differences and similarities for analysis .

3. Fascioliasis
 The species of genus Fasciola are the common liver flukes
of a wide range of animals. Severe negative economic
impact due to fascioliasis has been estimated in ruminants.
 Commonly three Species are found-
 Fasciola hepatica
 Fasciola gigantica
 Fasciola jaksoni
 Advanced techniques for identification and determining the
phylogeny of parasites have been developed. Molecular
methods include microsatellites and the analysis of
ribosomal and mitochondrial DNA sequence markers.
Markers have been used in separating Fasciola gigantica
from F. hepatica in various hosts using the nuclear
ribosomal internal transcribed spacers (ITS) 1 and 2.

4. Common Molecular
Approaches

5. Markers for Fasciola species
Molecular studies of a variety of organisms, including
Fasciola, have been carried out based on following
markers -
1. Mitochondrial DNA (mtDNA)
2. Ribosomal DNA (rDNA)
1. Mitochondrial DNA markers :
Mitochondrial CO1 & ND1 has been used in genotypic
and species identification of Fasciola spp. They are
useful in determining genetic diversity of flukes.

6. Markers for Fasciola species
2. Ribosomal DNA (rDNA) markers :
The nuclear rDNA markers (ITS-1 and ITS-2) are located
between the 18S, 5.8S and 28S ribosomal RNA genes.
They are useful in the identification and confirmation of
Fasciola species. The ITS-1 marker has been useful in the
differentiation of F. hepatica and F. gigantica.

7. Common Molecular
Approaches
DNA probes:
Before the availability of PCR-based approaches, DNA
probes were the alternative choice for the genotypic
detection of Fasciola spp.
Sequence Related amplified Polymorphism (SRAP):
The SRAP technique is useful for revealing genetic
variability within and between F. hepatica and F.
gigantica.
Randomly amplified polymorphic DNA (RAPD):
RAPD is a useful technique for the identification and
differentiation of F. hepatica and F. gigantica.

8. Common PCR based assays
Over the last two decades, several PCR-based assays have been
developed for the accurate identification of Fasciola spp
including-
a) PCR-linked restriction fragment length polymorphism
(PCR-RFLP)
b) PCR-linked single-strand conformation polymorphism
(PCR-SSCP)
c) Fluorescence-based SSCP (F-PCR-SSCP)
PCR-RFLP:
This assay targeting a 618-bp sequence of the 28S rDNA was
developed for the differentiation between F. hepatica and F.
gigantica. A similar PCR-RFLP assay using restriction
endonucleases Hsp92 II and Rca I was developed to differentiate
between F. Hepatica & F. gigantica.

9. Specific PCR assays
Recently, several specific PCR assays have been developed to
differentiate and detect F. hepatica and F. gigantica infection.
 Specific PCR method based on the ITS-2 sequences
 Specific PCR assays, using two primer sets derived from RAPD
sequences
 Taq-Man based real-time PCR
 A multiplex PCR assay
 Loop-mediated isothermal amplification (LAMP) allows
amplification of target nucleic acids under isothermal
conditions with high sensitivity, specificity, rapidity and
precision

10. Specific PCR assays
Sampling: Liver flukes are collected from abattoirs that have
a large slaughter capacity.
DNA extraction: Approximately 25mg of each fluke usually
used for DNA extraction using a DNeasy® DNA Blood and
Tissue Kit (Qiagen).
Spectrophotometry: Spectrophotometry is used to quantify
the fluke DNA samples. One μl of double-deionised water
was used to zero a Nanodrop Spectrophotometer and 1μl
of AE buffer from Qiagen then used to calibrate the
instrument. One μl DNA solution was added and the
concentration determined.

11. Specific PCR assays
Polymerase chain reaction (PCR):
• The PCR amplifications for both markers are performed in 25μL volumes.
Each reaction contained 4μL of DNA, 11μL of sterile water, 8μL of TopTaq
master mix (QIAGEN) and 1μL of each primer per reaction.
• PCR should be performed in a thermocycler under the following
conditions: 94°C for 5 min (initial denaturation), followed by 40 cycles at
95°C, 1 min (denaturation), 1 min (annealing), 72°C, 1 min (extension), and
a final extension of 72°C for 7 min.
• The annealing temperature is 55°C for the ITS region and 59°C for the CO1
region.
• An aliquot (8μl) of the reaction products then electrophoresed through a
1% agarose gel in order to allow separation and isolation of the
amplification products. The amplified ITS and CO1 bands are selected by
their positions relative to the co-electrophoresed molecular weight
marker and excised from the gel. The excised bands are stored in a 1.5ml
microfuge tube at -20°C ready for sequencing.

12. Specific PCR assays
DNA sequencing: DNA fragments should be sequenced in the
forward and reverse directions using the primers used in the
initial amplification

13. Conclusions
Conventional methods of detection and
differentiation of Fasciola do not accurately reflect
the full diversity of Fasciola spp.
Molecular genetics studies over the past two
decades have added significantly to our
understanding of Fasciola taxonomy and genetics.
These knowledge contributed to the development of
advanced approaches for the accurate identification
and differentiation of Fasciola spp.

14. References
 S. Mas-Coma, M. D. Bargues, and M. A. Valero, “Fascioliasis and other
plant-borne trematode zoonoses.”
 S. Mas-Coma, M. A. Valero, and M. D. Bargues, “ChapternFasciola,
lymnaeids and human fascioliasis, with a global overview on disease
transmission, epidemiology, evolutionary genetics, molecular
epidemiology and control.”
 M. A. Valero,N.A.Darce,M.Panova, and S.Mas-Coma, “Relationships
between host species and morphometric patterns in Fasciola hepatica
adults and eggs from the northern Bolivian Altiplano hyperendemic
region.”
 K. Tamura, D. Peterson, N. Peterson, G. Stecher, M. Nei, and S. Kumar,
“MEGA5: molecular evolutionary genetics analysis using maximum
likelihood, evolutionary distance, and maximum parsimony methods.”
 W. Y. Huang, B. He, C. R. Wang, and X. Q. Zhu, “Characterisation of Fasciola
species from Mainland China by ITS-2 ribosomal DNA sequence.”

15. Thank You