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ORIGINAL PAPER
Morphological and molecular identification of two
Paragonimus spp., of which metacercariae concurrently
found in a land crab, Potamiscus tannanti, collected in Yenbai
Province, Vietnam
Pham Ngoc Doanh &Akio Shinohara &Yoichiro Horii &
Shigehisa Habe &Yukifumi Nawa &Dang Tat The &
Nguyen Thi Le
Received: 20 September 2006 /Accepted: 23 November 2006 / Published online: 6 January 2007
#Springer-Verlag 2007
Abstract Paragonimosis is an important food-borne zoo-
nosis especially in Asian countries. Among Paragonimus
species, Paragonimus westermani followed by P. skrjabini
complex are the major pathogens for human paragonimosis
in Asia. In addition, P. heterotremus is an important
pathogen in southern China and the Indochina Peninsula
and is the only proven species to cause human para-
gonimosis in Vietnam. During a recent survey in Yenbai
Province in northern Vietnam, we found small and large
types of Paragonimus metacercariae often concurrently in
mountainous crabs, Potamiscus tannanti. Adult worms
from those small and large metacercariae were obtained
separately by experimental infection in dogs and cats.
Morphological and molecular phylogenetic study based on
sequences of ITS2 and a part of CO1 genes were performed
for the identification of small and large metacercariae and
their adults. The results showed that small metacercariae
and their adults are completely identical with P. hetero-
tremus in morphology and molecular genetic profiles. In
contrast, large metacercariae and their adults have some
morphological similarities with P. skrjabini and P. harina-
sutai, but are unidentifiable from each other by morphology
alone. Molecular phylogenetic tree analyses on ITS2 and
CO1 genes revealed that large metacercariae and their
adults were grouped in the same clade and different from
any known Paragonimus species. Although they share the
same ancestor with P. skrjabini complex, their genetic
distance was considerably different from two other known
subspecies, P. skrjabini skrjabini and P. skrjabini miyazakii.
Our results provide a new insight on the phylogeny of the
genus Paragonimus.
Introduction
Paragonimosis is a subacute to chronic lung disease caused
by infection with lung flukes, genus Paragonimus, and is a
typical food-borne parasitic zoonosis (reviewed by Blair et
al. 1999b). In Asian countries, the disease is endemic
Parasitol Res (2007) 100:1075–1082
DOI 10.1007/s00436-006-0411-9
P. N. Doanh :D. T. The :N. T. Le
Department of Parasitology, Institute of Ecology and Biological
Resources, Vietnamese Academy of Science and Technology,
Hanoi, Vietnam
P. N. Doanh
e-mail: pndoanh@iebr.vast.ac.vn
A. Shinohara
Department of Bio-resources, Frontier Science Research Center,
University of Miyazaki,
Miyazaki, Japan
e-mail: akioshi@med.miyazaki-u.ac.jp
Y. Horii (*)
Veterinary Teaching Hospital and Parasitology,
Faculty of Agriculture, University of Miyazaki,
Gakuen-kibanadai-Nishi,
Miyazaki 889-2192, Japan
e-mail: horii@cc.miyazaki-u.ac.jp
S. Habe
Department of Parasitology,
Fukuoka University School of Medicine,
Fukuoka, Japan
e-mail: shabe@fukuoka-u.ac.jp
Y. Nawa
Division of Parasitology, Department of Infectious Diseases,
Faculty of Medicine, University of Miyazaki,
Miyazaki, Japan
e-mail: yukifuminawa@fc.miyazaki-u.ac.jp
especially in China, Korea, Japan, and Thailand where
people prefer to eat uncooked/undercooked freshwater
crabs (WHO/FAO 2004). In Vietnam, although the first
human case of paragonimosis was discovered as early as in
1906 (Monzel 1906), the disease just has been paid
attention for public health issue in about the last 10 years
(Vien et al. 1994; Vien 1997; Le et al. 1997a; De et al.
2001; Doanh et al. 2002,2005). Recent epidemiological
surveys in Vietnam revealed that paragonimosis is endemic
in northwestern mountainous area especially Laichau and
the neighboring provinces (Vien 1997; De et al. 2001;
Doanh et al. 2002,2005).
Among over 50 species of the genus Paragonimus (Blair
et al. 1999b), Paragonimus westermani is known as the
major pathogen for human paragonimosis in Asia followed
by P. skrjabini complex (Blair et al. 1999b). In addition, P.
heterotremus is distributed in southern China and the
Indochina Peninsula and is an important pathogen for
human paragonimosis in this area (Blair et al. 1999b). To
date, P. heterotremus is the only proven species to cause
human paragonimosis in Vietnam (Kino et al. 1995; Vien
1997; De et al. 2001; Doanh et al. 2005; Le et al. 2006). Its
metacercariae were found in freshwater crabs in at least
eight provinces (Le et al. 1997a; De et al. 2001; Doanh et
al. 2002,2005; WHO/FAO 2004; Le et al. 2006). Even by
molecular study, Paragonimus metacercariae and adults
collected in four endemic provinces, Laichau, Sonla,
Yenbai, and Hoabinh, were identified as P. heterotremus
(Le and De 2004; Le et al. 2003,2006). Although P.
westermani (Landmann et al. 1961; Blair et al. 1999b) and
P. ohirai (Le et al. 1997b) were reported to be found in
Vietnam (Doanh et al. 2005), their morphological identifi-
cation is questionable, and their distribution remains
unclear (Le et al. 2006).
The aim of this study was, therefore, to see whether
Paragonimus species other than P. heterotremus are
detected in freshwater crabs, Potamiscus tannanti,in
Yenbai Province because people in this region prefer to
eat them in uncooked/undercooked condition, and this crab
is known to carry a heavy burden of P. heterotremus
metacercariae (Doanh et al. 2002).
Materials and methods
Materials
Metacercariae of Paragonimus were isolated from individ-
ual crabs, P. tannanti, collected from an endemic area of
Yenbai Province. In brief, the gills, liver, muscles, and other
organs of crabs were dissected into small pieces, and each
piece was pressed between two slide glasses and checked
carefully under a dissecting microscope. Freshly isolated
metacercariae were used for morphological observation and
experimental infection. For molecular study, metacercariae
were separately fixed in 70% ethanol and stored at −20°C
until use. Dogs and cats of 2 to 3-month-old were
purchased from a breeder in Hanoi where paragonimosis
is not endemic. All animals were examined for Para-
gonimus or any other parasite eggs by repeated fecal
examinations, and parasite-free animals only were used
for experiments. Seven dogs and two cats were infected
with small metacercariae, and two dogs and two cats with
large metacercariae. Each animal was orally given 10–20
alive metacercariae, and the appearance of Paragonimus
eggs in feces was monitored from 1 month thereafter
infection. Adult worms were collected from the lungs of
fecal egg-positive animals at necropsy. All adult worms
wereintensivelywashedinphosphate-bufferedsaline.
Then, some of them were immediately fixed in 70%
ethanol and kept at −20°C for extraction of genomic
DNA. For morphology, adult worms were flattened
between slide glasses and fixed in 70% ethanol. They were
then stained with carmine, cleared in xylene, mounted
individually onto a slide glass with Canada balsam, and
covered with a coverslip for permanent preparation.
Morphological study
Freshly isolated metacercariae and the permanent prepara-
tion of adult worms were examined under a light
microscope. The shape, size and cyst wall structure, and
the position of metacercaria in the cyst are observed
without coverslip, and these data are used for characteriza-
tion of metacercariae. For adult worms, the shape and the
size of body and organs, ratio of sucker sizes, arrangement
of cuticular spines, the size and the degree of branching of
ovary and testes, as well as egg’s shape and size are
employed as the morphological parameters.
Molecular analyses
The samples used for molecular analyses in this study are
listed in Table 1. Genomic DNA from individual ethanol-
preserved adult worms and metacercariae was extracted by
proteinase K digestion and phenol/chloroform/isoamyl
alcohol extraction procedures, followed by ethanol precip-
itation for purifying extracted DNA. The nuclear ribosomal
second internal transcribed spacer region (ITS2) was
amplified using the primer pair of 3S (5′-GGTACCGGTG
GATCACTCGGCTCGTG-3′)andBD2(5′-TATGCT
TAAATTCAGCGGGT-3′) described by Bowles and
McManus (1993) and Bowles et al. (1993). An amplifica-
tion of partial mitochondrial cytochrome oxidase subunit 1
gene (CO1) was obtained using primer pair of JB3 (5′-
TTTTTTGGGCATCCTGAGGTTTAT-3′) and JB4.5 (5′-
1076 Parasitol Res (2007) 100:1075–1082
TAAAGAAAGAACATAATGAAAATG-3′) also previous-
ly described by Bowles et al. (1993). The polymerase chain
reaction (PCR) products were purified using Qiaquick PCR
purification kit (Qiagen), primed using Big-Dye terminator
cycle sequencing kit v3.1 (ABI), and both strands directly
sequenced (Model 310 or 3100, Applied Biosystems).
Phylogenetic analyses
Eleven and nine nucleotide sequences of the ITS2 and CO1
genes of the samples, respectively, were determined
(Table 1). These sequences were deposited in the Gen-
Bank/EMBL/DDBJ nucleotide sequence database with
accession No. AB270675–AB270694. In addition, 18 and
19 sequences of ITS2 and CO1 gene of several Para-
gonimus species were obtained from the previous reports
(Blair et al. 1997,1999a; Iwagami et al. 2003; Blair et al.
2005) and DNA database (see Appendix 1). In this study,
we used Euparagonimus cenocopiosus as an outgroup
because we are not able to find out another proper CO1
and ITS2 sequence information of the species to be used as
the outgroup for Paragonimus in the superfamily Troglo-
trematoidea. Euparagonimus is morphologically classified
as a distinct genus (Chen 1965) and can be placed as sister
taxa to all Paragonimus species (Blair et al. 1999a),
although the taxonomic status of genus Euparagonimus is
still unclear (Blair et al. 1999a). Two sequence data sets,
ITS2 and CO1, were set up for reconstructing the
phylogenetic trees together with those sequences obtained
from DNA data bank and our experimental data in this
study. They were aligned using Clustal-X v1.83 (Thompson
et al. 1997) with default options. For each data set,
neighbor-joining (Saitou and Nei 1987) trees were con-
structed with the Kimura-2-parameter model (Kimura 1980)
using computer software program package PAUP* 4.0
(Swofford 2001). Finally, the statistical confidence of
branching patterns was evaluated by the bootstrap test
(Felsenstein 1985).
Results
Morphological data
Two types of Paragonimus metacercariae, small and large
ones, were collected from the same crabs of P. tannanti in
Yenbai Province. The small metacercariae were prevalent
and found in the gills, livers, and muscles of crabs, whereas
the large ones were less frequent and mainly found in the
livers of crabs. They are different in size and morphology
(Fig. 1).
The small metacercariae are 176–250×168–230 μm
(mean 212±18×187± 16 μm based on 30 metacercariae)
Table 1 Samples used in this study for molecular analyses
Sample code DNA extracted from Host details
a
Morphological size of metacercaria
b
Gene bank accession no.
ITS2 CO1
BM-YB1 Metacercaria Crab Potamiscus tannanti Large AB270692 AB270681
BM-YB2 Metacercaria Crab P. tannanti Large AB270693 AB270682
BM-YB3 Metacercaria Crab P. tannanti Large AB270694 AB270683
AD-P.sp.1 Adult worm Cat; fed with metacercariae Large AB270689 AB270678
AD-P.sp.2 Adult worm Dog; fed with metacercariae Large AB270690 AB270679
AD-P.sp.3 Adult worm Dog; fed with metacercariae Large AB270691 AB270680
SM-YB1 Metacercaria Crab P. tannanti Small AB270684 –
SM-YB2 Metacercaria Crab P. tannanti Small AB270685 –
SM-YB3 Metacercaria Crab P. tannanti Small AB270686 AB270675
AD-Ph.4 Adult worm Dog; fed with metacercariae Small AB270687 AB270676
AD-Ph.5 Adult worm Dog; fed with metacercariae Small AB270688 AB270677
a
All metacercaria were collected from Anlac commune, Lucyen district of Yenbai Province, Vietnam.
b
The morphological difference of metacercariae are shown in Fig. 1.
Fig. 1 Two types of Paragonimus metacercariae found in same crabs
(P. tannanti) collected from Yenbai Province. aSmall metacercaria (P.
heterotremus) and blarge metacercaria (Paragonimus sp.). Scale bar
100 μm
Parasitol Res (2007) 100:1075–1082 1077
in size having a thick inner cyst wall and apparently
identified as metacercariae of P. heterotremus. Adult worms
derived from small metacercariae were obtained from
experimentally infected dogs and cats 45–60 days after
infection. The prominent morphological characteristics are
single arrangement of cuticular spines and the oral sucker
(840×980 μm in average) nearly two times larger than the
ventral sucker (480 μm in diameter; Fig. 2a). These figures
are compatible with those of P. heterotremus (Chen and
Hsia 1964).
In contrast, the large metacercariae, which were found
for the first time in Vietnam, are provided with a thin inner
cyst wall, metacercaria occupies the entire space of the cyst
and measuring 738–943×648–931 μm (mean 811±79×
775±100 based on seven metacercariae). This size is bigger
than that of any other known Paragonimus species in Asia,
i.e., P. westermani (300–400 μm), P. skrjabini (420–
550 μm), and P. harinasutai (500–600 μm). Apart from
extremely large size, morphological features of large
metacercariae resemble those of P. skrjabini. After exper-
imental infection with large metacercariae in dogs and cats,
eggs became detectable in feces at around 90 days of
infection, and adult worms were successfully obtained from
the lungs of infected animals. Eggs are oval, 80–103×45–
57 μm (average 93±5×54 ± 3 μm) in size, and have clear
operculum at one end. The body of adult worms (Fig. 2b)
are measuring 9.4–10.0×5.0–5.2 mm. Cuticular spines are
singly arranged on the body surface. Oral sucker is 754–
760×738–750 μm (average 756 × 745 μm). Ventral sucker
is 810–836×894–902 μm (average 822 × 899 μm) and
situated in the middle of the body. The ventral/oral sucker
ratio is 1.2 (based on three samples). Vitelline glands
widely distribute on both sides of body. Testes are branched
and 864–884×1020–1312 μm (average 876× 1145 μm) in
size. Ovary is 800–820×1020–1230 μm (average 810 ×
1117 μm) in size and finely branched coral shape. Based on
the ratio of oral and ventral suckers, the adult worms
derived from large metacercariae are easily distinguished
from P. heterotremus, which has an oral sucker nearly twice
larger than ventral one. The adult worms grown from large
metacercariae share some morphological characteristics,
such as arrangement of cuticular spines and the size and
the ratio of oral and ventral suckers, with P. skrjabini
complex (Blair et al. 2005)andP. harinasutai (Miyazaki
and Vajrasthira 1967), but are difficult to distinguish from
each other merely by morphology.
Fig. 2 Adult worms of two
Paragonimus species recovered
from dog fed the small meta-
cercariae (aP. heterotremus) and
large metacercariae (bParago-
nimus sp.). Scale bar 1mm
1078 Parasitol Res (2007) 100:1075–1082
Molecular analysis
A total of 11 new samples of Paragonimus from Yenbai
Province were used in molecular phylogenetic analyses,
including three each of small and large metacercariae, two
adult worms developed from small metacercariae, and three
adult worms developed from large metacercariae. As a
result, we successfully determined 11 ITS2 and nine CO1
gene sequences from those Paragonimus samples (Table 1).
No insertions or deletions were found in CO1 gene data sets
(387 bp); however, sequence length of ITS2 gene varied
among samples (358–360 bp). The sequences were aligned
with those of several related species within genus Para-
gonimus obtained from DNA database (GenBank/EMBL/
DDBJ; Appendix 1) and constructed the phylogenetic trees
separately in ITS2 (Fig. 3) and CO1 sequence data set
(Fig. 4).
As was expected from morphological identification,
small metacercariae and their adult worms were com-
pletely identical with P. heterotremus;therewasno
difference among these samples and other populations of
this species in China and Thailand on ITS2 phylogenetic
tree (Fig. 3). In addition, those were also clustered into
monophyletic clade with P. heterotremus from China,
Thailand, and Vietnam with 100% support values on CO1
data set (Fig. 4). Thus, together with morphological
features, small metacercariae and their adult worms are
indeed P. heterotremus.
On the other hand, large metacercariae and their adult
worms were not clustered with any other Paragonimus
species in both ITS2 and CO1 trees; they reconstructed new
distinct phylogenetic groups (Figs. 3and 4). These samples
are closest to P. skrjabini complex (also included P.
skrjabini miyazakii as re-classified from P. miyazakii; see
Blair et al. 2005) and P. hokuoensis in both trees (Fig. 3and
4). However, the bootstrap support values of this node are
not so high (43 and 49% in ITS2 and CO1 data set,
respectively; Figs. 3and 4). In comparison of ITS2 and
CO1 sequences, the genetic distances of large metacercariae
and their adults differ from P. skrjabini and P. hokuoensis
complex at 6.7 and 10.6% on average, respectively. These
figures are smaller than those of P. heterotremus (average
ITS2, 7.1% and CO1, 12.1%); P. harinasutai (average
ITS2, 9.3% and CO1, 13.6%); P. macrorchis (average ITS2,
10.7% and CO1, 12.5%); P. ohirai (average ITS2, 11.6%
and CO1, 14.7%); and P. westermani (average ITS2, 12.1%
and CO1, 21.9%). Thus, newly discovered large metacer-
cariae and their adult worms were grouped in a new clade
distinct from any other known Paragonimus species,
though they might be sister to, or within, P. skrjabini
complex.
Discussion
Paragonimosis is endemic in the mountainous area of
northern Vietnam, and the causative pathogen was identi-
fied as P. heterotremus (Kino et al. 1995; De et al. 2001;Le
et al. 1997a,b; Doanh et al. 2002,2005; Le et al. 2006).
Although two other Paragonimus species, P. westermani
AF159602
AF159601
AF159604 P. westermani (THA)
U96907 P. westermani (JPN )
AF538945 P. mexicanus (ECU)
AF538946 P. mexicanus (GTM)
AB244621 P. bangkokensis
AB248091 P. bangkokensis (THA)
AF159609 P. harinasutai (THA)
U96911 P. ohirai (JPN)
AF159608 P. macrorchis (THA)
AY618758 P. heterotremus (CHN)
AF159603 P. heterotremus (THA)
SM-YB1
SM-YB2
SM-YB3
AD-Ph4
AD-Ph5
AY618756 P. hokuoensis (CHN)
AY618757 P. skrjabini miyazakii (JPN)
AY618740 P. skrjabini miyazakii (CHN)
AY618729 P. skrjabini skrjabini (CHN)
AY618751 P. skrjabini skrjabini (CHN)
AY618731 P. skrjabini skrjabini (CHN )
AY618734 P. skrjabini skrjabini (CHN)
AD-Psp1
AD-Psp2
AD-Psp3
BM-YB3
BM-YB2
BM-YB1
0.01 substitutions/site
ITS2 Euparagonimus cenocopiosus
100
94
43
50
100
59
51
93
100 91
54
100
100
Small metacercariae
and their adults
from Yenbai, Vietnam
Large metacercariae
and their adults
from Yenbai, Vietnam
48
65
Fig. 3 The neighbor-joining
tree reconstructed from ITS2
gene sequences. Bootstrap
scores (percentages of 1,000
replications) are presented for
each node. Samples obtained the
nucleotide sequences in this
study are represented with sam-
ple code (see Table 1), and
others from DNA database are
shown with accession no., spe-
cies name, and country code
(see also Appendix 1)
Parasitol Res (2007) 100:1075–1082 1079
(Landmann et al. 1961) and P. ohirai (Le et al. 1997b),
were reported to be found in Vietnam (Doanh et al. 2005),
their identification remains open (Le et al. 2006). The
present results clearly showed that in addition to P.
heterotremus, another Paragonimus species is endemic in
northern Vietnam.
In the present study, small metacercariae and their adults
were morphologically identical with those of P. hetero-
tremus, and their molecular phylogenetic analysis by CO1
and ITS genes clearly confirmed the results of morpholog-
ical identification. In contrast to easy and clear-cut
identification of small metacercariae and their adults as P.
heterotremus, we found difficulties for identification and
speciation of large metacercariae and their adult worms.
The size of large metacercariae is far bigger than that of any
other known Paragonimus species in Asia. The large
metacercariae examined in this study share some character-
istics with P. skrjabini metacercaria, such as having no
space in the cyst nor having pinkish globules in the cyst.
These are characteristics of P. harinasutai metacercaria
(Miyazaki and Vajrasthira 1967). Adult worms developed
from large metacercariae and those of P. skrjabini and P.
harinasutai share two prominent characteristics of having a
ventral sucker slightly larger than the oral one and single
arrangement of cuticular spines. The size of testes of the
adult worms developed from large metacercariae is similar
to that of P. harinasutai, but smaller than that of P.
skrjabini. As was discussed by Miyazaki and Vajrasthira
(1967), morphological identification of P. skrjabini and P.
harinasutai is difficult because their morphological charac-
teristics are very similar to each other. Although the size of
testes can be used to distinguish P. skrjabini and P.
harinasutai (Blair et al. 2005), it is variable and needs
statistical analysis. The number of adult worms of large
metacercariae is not sufficient to draw any conclusion for
speciation of this newly discovered Paragonimus species in
Vietnam by morphology alone.
The taxonomy of Paragonimus species has traditionally
relied on the morphology of metacercariae and adult
worms, and about 50 species of this genus have been
described based on the morphology (reviewed by Blair et
al. 1999b). However, some of them are indistinguishable
from each other by morphology and Blair et al. (1997,
1999b) put several species as the synonym of others.
Recently, mainly based on molecular phylogenetic analy-
ses, they divided Paragonimus species into five major
groups: P. westermani,P. ohirai,P. mexicanus,P. hetero-
tremus, and P. skrjabini (Blair et al. 2005). According to
their results, P. harinasutai is, together with P. bangkoken-
sis, close to P. ohirai and composed of a unique group
genetically different from other four groups. Regarding P.
skrjabini complex, they regard the following as synonyms
of P. skrjabini:P. miyazakii;P. szechuanensis;P. hueitun-
gensis; and P. veocularis. They also consider P. hokuoensis
within or sister to P. skrjabini. Interestingly, our result of
molecular analysis showed that newly discovered large
metacercariae and their adults genetically differ from any
other known Paragonimus species, although our specimen
share some morphological features with P. harinasutai and
P. skrjabini. All new samples reconstructed a new distinct
phylogenetic group closest to P. skrjabini and P. hokuoensis
complex in both trees with the bootstrap support values of
AF159594
AF159595
U97205 P. westermani (JPN)
U97213 P. westermani (PHL)
U97214 P. ohirai (JPN)
AF159600 P. harinasutai (THA)
0.01 substitutions/site
CO1 Euparagonimus cenocopiosus
AF159598 P. macrorchis (THA)
AY618836 P. hokuoensis (CHN)
AY618838 P. hokuoensis (CHN)
AY618762 P. skrjabini skrjabini (CHN)
AY618797 P. skrjabini skrjabini (CHN)
AY618783 P. skrjabini skrjabini (CHN)
AY618802 P. skrjabini skrjabini (CHN)
AY618806 P. skrjabini skrjabini (CHN)
AY618814 P. skrjabini miyazakii (JPN)
AY618832 P. skrjabini miyazakii (CHN)
AD-Psp1
BM-YB1
AD-Psp3
AD-Psp2
BM-YB2
BM-YB3
Large metacercariae
and their adults
from Yenbai, Vietnam
49
100
72
47
35
88
100
100
48
40
42
99
100
AF538944 P. mexicanus (GTM)
AF538936 P. mexicanus (ECU)
100
27 AY618841 P. heterotremus (VTN)
AF159597 P. heterotremus (THA)
AY618840 P. heterotremus (CHN)
SM-YB3
AD-Ph5
AD-Ph4
Small metacercariae
and their adults
from Yenbai, Vietnam
100
51
98
Fig. 4 The neighbor-joining
tree based on CO1 gene
sequences. Bootstrap scores
expressed as percentages of
1,000 replications are given at
each node. New sequences
obtained in this study are repre-
sented with sample code
(see Table 1), and others from
DNA database are indicated
with accession no., species
name, and country code
(see also Appendix 1)
1080 Parasitol Res (2007) 100:1075–1082
this node being 43 and 49% in ITS2 and CO1 data set,
respectively (Figs. 3and 4). Related to this point, Blair et
al. (2005) reported that the variance in ITS2 sequence is
very small within a species. In the present study, ITS2
sequences of large metacercariae and their adults were
closest to P. skrjabini at 6.7% genetic distances (see
“Results”), which seems to be slightly larger variance as an
intraspecific diversity, suggesting that this new group
would be classified as a new species. Together with
relatively low bootstrap values of the node of our new
samples and P. skrjabini complex, further study using
various Paragonimus samples collected in various localities
in Vietnam and other countries is necessary for the
clarification and the phylogenetic relationships of these
distinct groups.
In conclusion, present results clearly showed that, in
addition to P. heterotremus, another species Paragonimus
sp. is distributed in northern mountainous areas of Vietnam.
In spite of partial morphological similarities with P.
skrjabini and P. harinasutai either in metacercariae or
adults, the results of molecular phylogenetic analysis
suggest that the newly discovered Paragonimus sp. in the
Yenbai Province of Vietnam is sister to P. skrjabini
complex or reconstruct a new Paragonimus species.
Whether the newly discovered Paragonimus sp. would be
a pathogen for humans should be determined in future by
molecular analysis of Paragonimus eggs in sputum and/or
stool of patients in this province.
Acknowledgment One of the authors, Pham Ngoc Doanh, received
financial support from the Japanese Society for Promotion of Science
(JSPS) Ronpaku Fellowship No. NCST-10430 for Ph.D. degree. This
work was also supported in part by Korea Foundation for Advanced
Studies (KFAS) to Prof. Nguyen Thi Le. The authors also would like
to send special thanks to colleagues at the Department of Bio-
resources, Frontier Science Research Center, University of Miyazaki
for kindly giving the best conditions for conducting molecular study.
Appendix
Appendix 1: Accession numbers of sequences obtained
from DNA database for reconstructing the phylogenetic
trees
Sources of data: 1, Blair et al. (1999a); 2, Blair et al.
(1997); 3, Iwagami et al. (2003); 4, Blair et al. (2005); 5,
published only in database. Euparagonimus cenocopiosus
ITS2: AF159601–159602 (Blair et al. 1999a); CO1:
AF159594–159595 (Blair et al. 1999a). P. westermani
ITS2: AF159604 (Blair et al. 1999a) and U96907 (Blair
et al. 1997); CO1: U97205 and U97213 (Blair et al. 1997).
P. ohirai ITS2: U96911 (Blair et al. 1997); CO1: U97214
(Blair et al. 1997). P. harinasutai ITS2: AF159609 (Blair et
al. 1999a); CO1: AF159600 (Blair et al. 1999a). P.
mexicanus ITS2: AF538945–538946 (Iwagami et al.
2003); CO1: AF538944 and AF538936 (Iwagami et al.
2003). P. bangkokensis ITS2: AB244621 and AB248091
(published only in database). P. m a c r o rc h i s ITS2:
AF159608 (Blair et al. 1999a); CO1: AF159598 (Blair et
al. 1999a). P. heterotremus ITS2: AY618758 (Blair et al.
2005) and AF159603 (Blair et al. 1999a); CO1:
AY618840–618841 (Blair et al. 2005) and AD159597
(Blair et al. 1999a). P. hokuoensis ITS2: AY618756 (Blair
et al. 2005); CO1: AY618836 and AY618838 (Blair et al.
2005). P. skrjabiniskrjabini ITS2: AY618729, AY618731,
AY618734 and AY618751 (Blair et al. 2005); CO1:
AY608762, AY618797, AY618783, AY618802 and
AY618806 (Blair et al. 2005). P. skrjabini miyazakii ITS2:
AY618740 and AY618757 (Blair et al. 2005); CO1:
AY618814 and AY618832 (Blair et al. 2005).
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