Human fascioliasis and the presence of hybridintrogressed forms of fasciola hepatica and fasciola gigantica in vietnam

Human fascioliasis and the presence of hybrid/introgressed formsof Fasciola hepatica and Fasciola gigantica in Vietnam aDepartment of Immunology, Institute of Biotechnology, Hanoi, Viet

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Human fascioliasis and the presence of hybrid/introgressed forms

of Fasciola hepatica and Fasciola gigantica in Vietnam

aDepartment of Immunology, Institute of Biotechnology, Hanoi, Viet Nam

bDepartment of Parasitology, Hanoi Medical University, Viet Nam

cDepartment of Environmental Health Sciences, Kochi Medical School, Kochi, Japan

dNational Institute of Veterinary Research, Hanoi, Viet Nam

eQueensland Institute of Medical Research, Brisbane, Qld 4006, Australia

fSchool of Marine and Tropical Biology, James Cook University, Townsville, Qld 4811, Australia

Received 19 June 2007; received in revised form 1 October 2007; accepted 2 October 2007

Abstract

The two species common of liver ﬂuke, Fasciola hepatica and Fasciola gigantica, cause human fascioliasis Hybrids between these spe-cies, and introgressed forms of Fasciola, are known from temperate and subtropical regions of eastern Asia Here, we report the presence

of hybrid and/or introgressed liver ﬂukes in Vietnam where it has recently been recognised that human fascioliasis is an important zoo-notic disease Specimens examined came from domestic stock (cattle and buﬀalo) at slaughter and also from human patients DNA sequences were obtained from the nuclear ribosomal second internal transcribed spacer (ITS-2) and from portions of two mitochondrial

protein-coding genes Mitochondrial sequences in every case were similar to those of Fasciola gigantica Nuclear ITS-2 sequences belonged to one or other of the Fasciola species, or, sequences from both were found in the same individual worm This study extends the known range of hybrids or introgressed forms of Fasciola into tropical regions of Asia.

Keywords: Fasciola gigantica; Fasciola hepatica; Hybridisation; Internal transcribed spacer region; Introgression; Vietnam

1 Introduction

The commonest and most widespread liver ﬂukes of the

genus Fasciola are Fasciola hepatica Linnaeus, 1758 (mostly

in temperate regions) and Fasciola gigantica Cobbold, 1856

(mostly tropical in distribution) Adults of both species occur

in many domestic ruminants and in humans (Mas-Coma

et al., 2005; Le et al., 2007) and can cause serious disease

The two liver ﬂuke species appear to be sympatric in many

subtropical and warm temperate areas, especially in Africa

and Asia (Le et al., 2007) They can generally be

et al., 2006), but the existence of individuals with intermedi-ate morphological characteristics can cause confusion (e.g Terasaki et al., 1982; Itagaki et al., 2005a) and has led to

et al., 2002) or morphometric methods (e.g.Ashraﬁ et al., 2006) to distinguish between the species It is desirable to know which species is the agent of human or animal disease

in a given area The two species differ in pathological and epi-demiological characteristics (Mas-Coma et al., 2005) Difficulties in specific identification have been most intensively studied in Japan and adjacent areas Research there has revealed not only a confusing range of morpho-logical forms but also the presence of worms of different ploidies (diploid, triploid and ‘‘mixoploid’’), all of which are parthenogenetic and do not produce normal sperm (Terasaki et al., 1982, 2000) Genetic studies on Japanese

doi:10.1016/j.ijpara.2007.10.003

* Corresponding author Tel.: +61 7 4781 4322; fax: +61 7 4725 1570.

E-mail address:david.blair@jcu.edu.au (D Blair).

www.elsevier.com/locate/ijpara International Journal for Parasitology 38 (2008) 725–730

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and Korean worm populations have detected individuals

that have both nuclear and mitochondrial sequences

typi-cal of F hepatica and others that appear on the same

grounds to be F gigantica Individuals also occur that

resemble one species in their nuclear DNA (usually assayed

using ribosomal RNA gene or spacer sequences) but have a

mitochondrial genotype typical of the other species

(Agat-suma et al., 2000; Itagaki et al., 2005a,b) Individuals have

also been found that have, in their tandem array of nuclear

ribosomal genes, copies of genes apparently derived from

Huang et al., 2004 in north eastern China; Itagaki et al.,

An obvious conclusion is that hybridisation and

in Fasciola populations in this part of Asia, processes that

can lead to production of polyploid and parthenogenetic

individuals (Dowling and Secor, 1997) Such individuals

are often aspermic There have been no convincing

demon-strations of hybrid/introgressed liver ﬂuke populations

out-side Asia, although aspermic triploid individuals of ‘‘pure’’

2004) Examples of triploid Fasciola sp are also known

2000) and Vietnam (Itagaki et al., 2005a) In addition to

Japan and Korea, aspermic Fasciola spp of unknown

ploidy are present (but generally uncommon) in the

Philip-pines, Vietnam, Thailand, Taiwan, India, Nepal and

Hawaii (Terasaki et al., 1982)

In Vietnam, fascioliasis (caused by morphologically

identiﬁed F gigantica) is very common in cattle and water

buﬀaloes, with prevalences of more than 50% in the Red

River and Mekong deltas, as well as in other coastal

regions (Bui et al., 2003) In recent years, an extraordinary

number of human cases of fascioliasis has been reported, to

such an extent that this zoonotic infection has become a

major public health concern in Vietnam More than 500

human cases were recorded in the three years from 1997

to 2000 based on serological tests (Tran et al., 2001) Prior

to 1991, fascioliasis had been regarded as rare in Vietnam

For example, only two cases were reported in 1978 (Tran

et al., 2001) Unusual cases of cutaneous fascioliasis have

2007) Morphologically, the adult ﬂukes found in animals

and human patients in Vietnam fall into two categories,

one typical of F gigantica and the other closely resembling

F hepatica Here we report data indicating that

hybrid/int-rogressed populations of Fasciola occur in Vietnam and

that these are implicated in human infection

2 Materials and methods

2.1 Sources of Fasciola specimens

Parasite material and nucleotide sequences of Fasciola

species, their host and geographical origin used in this

study are listed inTable 1

Adult worms of Fasciola sp of Vietnamese origin,

col-lected during 2001–2005 from human patients and animals (cattle and buﬀaloes), were preserved in 70% ethanol and

Vietnamese samples, 12 were of human origin, of which two (FspN-VN and FspQB-VN) were from the cases involving unusual cutaneous migration of worms reported

inLe et al (2007) Specimens from eight human cases were obtained surgically In two further cases, eggs

morpholog-ically identiﬁed as being of a Fasciola sp were recovered

from faeces of human patients serologically positive for fascioliasis These eggs were allowed to develop and hatch

in water (1–2 weeks) and 10–20 miracidia from each patient were collected for subsequent DNA extraction

2.2 Genetic markers Genetic markers including mitochondrial genes (cox1, nad1) and the nuclear second internal ribosomal spacer

(ITS-2) sequences were obtained ITS-2 is a useful marker

for distinguishing between F gigantica and F hepatica It

is rather conserved, especially in F hepatica There are

seven sites at which the two species typically diﬀer One

of these is a deletion in F gigantica relative to F hepatica

that appears to be a diagnostic diﬀerence between the spe-cies (e.g.Adlard et al., 1993; Semyenova et al., 2005) There

is some variability, especially in F gigantica, that can be

confusing (Le et al., 2007) Sequence data from mitochon-drial genes are more variable than is the case for ITS-2, but also provide unambiguous recognition of the two species (Le et al., 2007)

2.3 DNA extraction, PCR and sequencing

Total genomic DNA was extracted from adult worms and miracidia using the commercial QIAamp DNA extrac-tion kit (QIAGEN Inc.) according to the manufacturer’s instructions In the case of adult worms, only a single spec-imen was used in each DNA extraction Genomic DNA was diluted to a working concentration of 50 ng/lL and

2 lL of this was used as template in a PCR reaction of

50 lL

PCR was used to amplify the entire nuclear ITS-2 and two mitochondrial genetic markers (a portion of each of

as previously published (Bowles and McManus, 1993;

nad1 were designed for use in this study.

PCR ampliﬁcation was carried out in a ﬁnal volume of

50 lL, including 100 ng template, 10 pmol of each primer

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and a mix of the remaining PCR components (PCR Master

Mix from Promega) The PCR reactions were carried out

in a MJ Thermal Cycler PTC-100 (MJ Research, USA)

for cox1 with initiation at 94 °C for 5 min, then 35 cycles

including denaturation at 94 °C for 1 min, annealing at

37 °C for 1 min, extension at 72 °C for 2 min For ITS-2

Table 1

List of Fasciola specimens and sequences used in this study, their host and geographical origins

cox1 nad1 ITS-2

EU260073

F gigantica C Asia FgSemyenovaG5, G6, G7, G8 Adult N/A p Semyenova et al., 2005

N/A: not available.

a

Indicates apparent hybrid/ introgressed specimens (see text).

b

Indicates two worms known to have ITS-2 sequences of both Fasciola hepatica and Fasciola gigantica type For Vietnamese worms, G = F gigantica – like and H = F hepatica - like.

c Indicates data from Agatsuma et al (2000)

d Indicates specimens that had undertaken cutaneous migration in humans as reported in Le et al (2007)

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and nad1 the above conditions were used, except that

annealing was at 50 °C for 1 min Finally, all reactions were

held for 10 min at 72 °C to complete the ampliﬁcation

The PCR products were puriﬁed using QIAquick

Puriﬁ-cation kit (QIAGEN) PCR products were initially

sub-jected to direct sequencing using the PCR primers as

sequencing primers However, when traces with

ambigui-ties were obtained (ITS-2), PCR products were cloned

using a TA cloning kit (Invitrogen, USA) and recombinant

plasmid DNAs subjected to sequencing using Big Dye

Ter-minator Cycle Sequencing technology on an automated

sequencer (ABI 3100 Avant Genetic Analyzer) using M13

forward and reverse primers

2.4 Data analysis and phylogenetic construction

The sequences were edited in SeqEd v 1.03, aligned

using AssemblyLIGN v 1.9c and analysed using the

Mac-Vector 8.2 package (Accelrys) in a Macintosh computer

system Specific identification was confirmed by

compari-son with known sequences of the corresponding species

in GenBank or by reference to our previous published data

Le et al., 2001; Le and Nguyen, 2002

The multiple alignment of sequences was performed

using GeneDoc v2.5 (Nicholas and Nicholas, 1997

Gene-Doc: A tool for editing and annotating multiple sequence

alignment Distributed by author) and/or ClustalW

incor-porated into the MacVector 8.2 package Modeltest v3.7

(Posada and Crandall, 1998) was used to ﬁnd the best

sub-stitution model This model (HKY+G) was then speciﬁed

for likelihood analyses in PAUP* v4.0b10 (Swoﬀord, D

L 2000 PAUP* Phylogenetic Analysis Using Parsimony

(*and Other Methods) Version 4 Sinauer Associates,

Sun-derland, Massachusetts) PaupUP graphical interface

(Cal-endini, F., Martin J.-F 2005 PaupUP v1.0.3.1 A free

graphical frontend for PAUP* Dos software Available

JFM/PaupUp/main.htm) was used to facilitate working

with PAUP* The dataset was resampled 100 times using

the bootstrap method Phylogenetic analyses are presented

(sequences from Fascioloides magna DQ683545 and

Fasci-olopsis buski DQ341852) were used in some analyses to

conﬁrm monophyly of each Fasciola species.

As far as possible, all available published ITS-2, cox1

and nad1 sequences have been included Some sequences

have been omitted – this is especially so for ITS-2 in which

there are relatively few variable sites Sequences with

ambiguous sites or incomplete sequences can bias the tree

dramatically Available ITS-2 sequences not used were:

AB207153 (Hiroshima and Kagoshima, Japan), which

includes several ambiguous sites reﬂecting the fact that

ITS-2 sequences of both Fasciola species are present

(Itag-aki et al., 2005a); AB259058 (Kyoto, Japan), which is

incomplete; L07844, one of the earliest reported sequences

of F hepatica, which has possible sequencing errors at the

which has many probable errors near the 50end Sequences reported byAdlard et al (1993)(F gigantica from Indone-sia and MalayIndone-sia; F hepatica from Australia, New Zea-land, Hungary and Mexico; Fasciola sp from Japan) are

shorter than those presented here and have not been included Their inclusion would not have altered the

near the 30end that are inverted relative to all other avail-able sequences However, Prof Zhu Xing-Quan (personal communication), one of the authors of that paper, has con-ﬁrmed that this inversion is a sequencing error: their sequences have been corrected as used in this paper (note

sequences) Sequences for some haplotypes from Turkmen-istan and adjacent regions of West and Central Asia

recon-structed from information in that paper: none was avail-able from any database Immediately prior to submission

of this paper, a further sequence of F gigantica, from

Meghalaya, India, appeared in the public databases This

is EF027103, identical in sequence to the Indonesian

refer-ence sequrefer-ence of F gigantica mentioned below Sequrefer-ences

of F hepatica from Bolivia (Mas-Coma et al., 2001) are

identical to those from Spain

For reference, F gigantica from Indonesia (Fg_ID) and

F hepatica from Australia and Europe (Fh_AU) are

regarded as representing genetically pure forms of each species that have not experienced introgression or hybrid-isation (Agatsuma et al., 2000) (indicated by arrows on Fig 1)

3 Results Excluding ﬂanking regions in the 5.8S and 28S genes, the length of the ITS-2 alignment was 362 bp For the mito-chondrial markers, partial sequences were obtained from

cox1 (423 bp) and nad1 (435 bp).

The main purpose of the phylogenetic analyses was to compare species-of-origin of nuclear and mitochondrial sequences in a number of individual worms from Vietnam and elsewhere This is easily done by presenting a tree based on one marker on which can be indicated, for each individual, species-of-origin of the other marker(s) Here

we present only the tree of the ITS-2 sequences (Fig 1) Given the small number of diﬀerences between the species

in the ITS-2 region, and the slight variation within each species, it is not surprising that bootstrap values are rather low However, the two species are clearly distinguished (and remain so in analyses using other fasciolids as out-groups) It is immediately apparent that the Vietnamese

worms fall readily into the F gigantica or the F hepatica

cluster based on ITS-2 data However, all Vietnamese

worms had mitochondrial sequences (both cox1 and nad1) typical of F gigantica only Mitochondrial genomes

in animals are maternally inherited Our data thus demon-strate that hybridisation or introgression has occurred

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involving the two Fasciola species and that F gigantica was

the maternal parent in each case

Direct sequencing of PCR products from some of the

Vietnamese worms yielded traces with ambiguities

indicat-ing the presence of both F hepatica and F gigantica ITS-2

sequences (data not shown) Clean sequence for only one

type of ITS-2 was subsequently obtained from cloned

PCR products, which is why each of these worms is

included in only one clade inFig 1

All ITS-2 sequences (except one) from populations of

either species that, because of their geographical origins,

had probably not experienced introgression/hybridisation, were placed where expected on the tree (Fig 1) However, for many of these worms, ITS-2 only was available and nothing was known about their mitochondrial genome

The exceptional sequence was from a specimen of F gigan-tica from Zambia (AB010975) This sequence had the sin-gle-base deletion characteristic of F gigantica, but at

most other variable sites it possessed the base typical of

F hepatica We are uncertain how to interpret this.

Individual worms from human patients were distributed

in both clades of the tree (Fig 1)

4 Discussion

It is clear that hybridisation and/or introgression has occurred in Vietnam involving both of the common species

of Fasciola In every case in that country, the mitochon-drial genome was typical of F gigantica, whereas the

nuclear genome could be from either species Distinguish-ing between hybridisation and introgression requires more

oﬀspring of a mating between the two species In such a case, all oﬀspring will carry the mitochondrial genome of the maternal parent However, their nuclear ribosomal RNA cluster will contain copies from both parents Back-crossing of hybrids and of subsequent generations with one parent species (=introgression) should homoge-nise the ribosomal array so that sequences of the other

non-recombining nature of the mitochondrial genome means that it will be passed on largely unchanged from generation

to generation If back-crossing of hybrids is with the pater-nal species, then the mitochondrial genome of the materpater-nal species will introgress into the paternal species (reviewed in Blair, 2005) All this assumes that the organisms involved reproduce sexually and undergo normal meiosis For many

eastern Asian populations of Fasciola species, this

assump-tion seems to be false If hybrids are parthenogenetic, then worms should carry ribosomal repeats from both parents,

as well as the mitochondrial genome of the maternal par-ent, for as many generations as the clonal lineage persists

We do not yet know the ploidy of any of the Vietnamese worms, nor do we know if any are parthenogenetic How-ever, in at least some cases, individual worms carried ribo-somal sequences from both parent species, suggesting

introgression, or that these worms were from parthenoge-netic lineages of unknown age

Not all human infections are due to hybrid/introgressed

worms Some were apparently due to ‘‘pure’’ F gigantica

infection In addition, worms from human patients were scattered in both clades in the tree (Fig 1), suggesting that

multiple genotypes of Fasciola in Vietnam are capable of

infecting humans We have no information on whether

the mixing of genetic material of the two Fasciola species

in Vietnam is a recent phenomenon Nor do we know if

‘‘pure’’ F hepatica is present in the country.

Fig 1 Tree inferred from ITS-2 sequences Specimen codes are those used

in Table 1 GenBank accession numbers are included where available.

Sequences from human cases are shown in bold-italic type Arrows point

to reference sequences from ‘‘pure’’ Fasciola gigantica or Fasciola hepatica.

With the few exceptions discussed in the text, sequences fall into two

groups corresponding to F gigantica and F hepatica Sequences are

labeled according to the species name or code given to them in the relevant

original publications: Fg = F gigantica, Fh = F hepatica and Fsp for

those not assigned to a species Specimens from Vietnam are indicated by

the letters ‘‘VN’’ after the specimen code Other country codes: AR,

Armenia; AT, Austria; AU, Australia; BF, Burkina Faso; BV, Bolivia;

CA, Turkmenistan and adjacent regions of Central Asia; CN, China; FR,

France; ID, Indonesia; IN, India; IR, Ireland; JP, Japan; Kr, Korea; RU,

Russia, and neighbouring countries; SP, Spain; TL, Thailand; UR,

Uruguay; ZA, Zambia Specimens known to have F gigantica-like

mitochondrial sequences (this study or from the literature) are indicated

by * Similarly, specimens with F hepatica-like mitochondrial sequences

are indicated with # after the specimen code Neither symbol appears if

mitochondrial data are not available for that individual The two

sequences marked with Ù came from the same worm specimen in north

eastern China ( Huang et al., 2004 ) The symbol à indicates worms from

Vietnam for which direct-sequencing traces indicated the presence of both

F hepatica and F gigantica ITS-2 sequences Clean sequence for only one

type of ITS-2 was subsequently obtained from cloned PCR products,

which is why the worms are included in only one clade Bootstrap values

(as percentage support for each branch) are shown.

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In conclusion, we have demonstrated the presence in

Vietnam of hybrid and/or introgressed populations of liver

ﬂukes bearing genetic material from both F hepatica and

F gigantica Some of these worms were from human

patients This appears to be the ﬁrst demonstration from

a tropical country of the presence of liver ﬂukes containing

genetic material from both common species of Fasciola.

Acknowledgments

This investigation received ﬁnancial support from

IC-GEB (Project No CRP/VIE05-02 awarded to Thanh

Hoa Le) and the Wellcome Trust (Grant No 068762

awarded to Le, McManus and Blair) We thank our

collab-orators for their kind provision of materials used in this

study and staﬀ in Dr Le’s Laboratory (Institute of

Biotech-nology, Hanoi, Vietnam) for their laboratory work

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