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PHD : Parasites, Hosts and Diseases

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Original Article

Distribution Status of Hybrid Types in Large Liver Flukes, Fasciola Species (Digenea: Fasciolidae), from Ruminants and Humans in Vietnam

The Korean Journal of Parasitology 2018;56(5):453-461.
Published online: October 31, 2018

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*Corresponding author (imibtvn@gmail.com)
• Received: September 2, 2018   • Revised: October 16, 2018   • Accepted: October 17, 2018

Copyright © 2018 by The Korean Society for Parasitology and Tropical Medicine

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Distribution Status of Hybrid Types in Large Liver Flukes, Fasciola Species (Digenea: Fasciolidae), from Ruminants and Humans in Vietnam
Korean J Parasitol. 2018;56(5):453-461.   Published online October 31, 2018
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Distribution Status of Hybrid Types in Large Liver Flukes, Fasciola Species (Digenea: Fasciolidae), from Ruminants and Humans in Vietnam
Korean J Parasitol. 2018;56(5):453-461.   Published online October 31, 2018
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Distribution Status of Hybrid Types in Large Liver Flukes, Fasciola Species (Digenea: Fasciolidae), from Ruminants and Humans in Vietnam
Image Image
Fig. 1 Representative adult individuals sorted out for groups of small (S), medium (M) and large (L) shaped Fasciola liver flukes based on morphological examination by their size and the inferred ratio (BL/BW) between the body length (BL) and body width (BW). Size is indicated by a bar (1.2 cm). (A) fresh; (B) preserved flukes.
Fig. 2 Phylogenetic tree showing topology of haplogroup relationships of Fasciola spp. from Vietnam and the reference isolates/strains from GenBank (Table 3) based on analysis of partial mitochondrial nad1 nucleotide sequence data (535 nucleotides) using Paragonimus westermani as an outgroup. Phylogenetic tree reconstruction was performed by MEGA 7.0 using a maximum likelihood (ML) analysis based on the general time-reversible model; supported for each node by 1,000 bootstrap resamplings [32]. In each sequence, species abbreviation is followed-up by strain and country (where the fluke was isolated). Names of haplogroups are included for reference (in brackets). Accession numbers are given at the end of each sequence. Basal node for F. gigantica is indicated by an arrow. Fasciola spp. sequences of this study from Vietnam are indicated by solid circles. Fsp: Fasciola species indicating intermediate forms (admixed/introgressive); number in bracket: identical nad1 sequences obtained in this study.
Distribution Status of Hybrid Types in Large Liver Flukes, Fasciola Species (Digenea: Fasciolidae), from Ruminants and Humans in Vietnam

No. of Fasciola samples collected from Northern and Central Provinces in Vietnam from ruminants and humans

Provinces Ruminants Humans
1 Cao Bang 95
2 Lang Son 121
3 Bac Kan 56
4 Yen Bai 98
5 Thai Nguyen 2,170
6 Ha Noi 772 6
7 Hung Yen 135
8 Hai Duong 88
9 Ninh Binh 218
10 Nghe An 94 1
11 Ha Tinh 66
12 Thua Thien-Hue 123 1
13 Quang Nam 36 1
14 Binh Dinh 125 3
15 Phu Yen 104
16 Khanh Hoa 188 1
17 Dak Lak 79
18 Ninh Thuan 157
19 Quang Binh 1
Total 4,725 14

Primer sequences used for amplification and sequencing internal transcribed spacers (ITS) and mitochondrial nad1 gene for molecular discrimination of Fasciola species

Genomic origin Regions/Genes used in this study Forward Reverse Length of amplicons (bp)


Name Sequence (5′ to 3′) Name Sequence (5′ to 3′)
Nuclear ribosomal transcription unit (rTU or rDNA) ITS1(421 bp) U18S2F TCGTGACTGGGATCGGGGC U3SR CGACCCTCGGACAGGCG 852
ITS1-5.8S-ITS2 U18S2F TCGTGACTGGGATCGGGGC U28S2R GACCTTGGAGTCGGGTTGT 1,519–1,520
ITS2 (361–362 bp) U3SF CGGTGGATCACTCGGCTCGTG U28S2R GACCTTGGAGTCGGGTTGT 811–812

Mitochondrial genome (mtDNA) *Partial nad1(535 bp) FANDF AGATGTGTGCTCTGCGAGC FANDR GAGTTGRCTGGCCGGTA 1,170

F, Forward; R, Reverse.

*A partial sequence (535 bp) of nad1 gene was used for haplogroup analysis.

List of nad1 reference sequences and their accession numbers from GenBank and published database used for phylogenetic analysis of F. gigantica haplogroups in the present study

Species Country/Accession number References and GenBank
Fasciola gigantica

Haplogroups*
 Haplogroup A Bangladesh (AB894370); India (LC012900; LC128314); Nepal (AB894337; AB894338); Myanmar (AB604022); [6,7,16,24,28,29,36,38]
GenBank
 Haplogroup B India (LC012897; LC012899); Myanmar (AB604007); Thailand (AB603724);
 Haplogroup C China (AB477364; AB477369); Indonesia (LC127274; LC127264); Vietnam (AB385616);
 Haplogroup D Egypt (LC076199; LC076204; AB554156; AB554167; AB554194);
 Haplogroup E Zambia (AB983823; AB983824; AB983832; AB983833; AB983835)
 Unclassified China (AB477368; AB604941; AB604939; AB604932); Myanmar (AB604020); Japan (AB207168); Korea (AB211240); Vietnam (MF430851; MF430852; MF430854; AB385619; AB536756; MF430855; MF287790; AB385617; MF430855); GenBank
Fasciola hepatica Australia (AF216697); China (AB477359); Egypt (AB554179); Ireland (AB207156); Peru (LC070666); Spain (KF111652); Uruguay (AB207154) [7,17,23,33,39]

Paragonimus westermani Korea (AF219379) GenBank

*Haplogroups suggested by Amer et al. [7].

No. of small (S), medium (M) and large (L) individual shapes based on morphological examination of flukes present in the Fasciola spp. population (n=4,725 adults) from ruminants

Physical examination No. Size (cm) Inferred ratio (BL/BW) %
S (Small) (n=284) 2.0–3.6×1.3 1.54–2.77/1 6
M (Medium) (n=614) 3.6–4.2×1.3 2.77–3.23/1 13
L (Large) (n=3,827) 4.2–6.5×1.2 3.23–5.4/1 81
Total (n=4,725) 100

BL, body length; BW, body width.

Molecular delineation of Fasciola species and types of hybridization among S-, M- and L-shaped liver flukes from ruminants and human samples in Vietnam based on the key nucleotide markers of ITS1 and ITS2 and mitochondrial nad1

ITS1 ITS2 rDNA status No. of liver flukes from ruminants mtDNA status (nad1) Delineation of Fasciola spp. in ruminants No. of samples from humans (%)



17 107 201 279 299* 327** L (n=40) (%) M (n=40) (%) S (n=40) (%)
T T T A T
RsaI(−)
- Fgig 40/(100) 31 (77.5) 0 Fgig F. gigantica (n=71) 9 (64.0)

C A C T C
RsaI(+)
T Fhep 0 6 (15.0) 36 (90.0) Fgig Introgressive Fasciola sp. (n=42) 3 (22.0)

Y W Y W Y
RsaI(+/−)
-/T Mixed (Fhep/Fgig) 0 3 (7.5) 4 (10.0) Fgig Admixed Fasciola sp. (n=7) 2 (14.0)

ITS1, ITS2, internal transcribed spacer 1 and 2; Fhep, F. hepatica; F. gigantica; rDNA, ribosomal DNA; mtDNA, mitochondrial DNA; S, small; M, medium, L, large shape; Y, T or C; W, T or A.

*299: F. hepatica possesses GTAC-sequence for a RsaI(+) at positions 296–299 in ITS1, while F. gigantica does not (GTAT/(RsaI(−));

**327: At position 327 in ITS2, a nucleotide T (Thymine) present in F. hepatica and absent in F. gigantica [11,21,37].

Table 1 No. of Fasciola samples collected from Northern and Central Provinces in Vietnam from ruminants and humans
Table 2 Primer sequences used for amplification and sequencing internal transcribed spacers (ITS) and mitochondrial nad1 gene for molecular discrimination of Fasciola species

F, Forward; R, Reverse.

A partial sequence (535 bp) of nad1 gene was used for haplogroup analysis.

Table 3 List of nad1 reference sequences and their accession numbers from GenBank and published database used for phylogenetic analysis of F. gigantica haplogroups in the present study

Haplogroups suggested by Amer et al. [7].

Table 4 No. of small (S), medium (M) and large (L) individual shapes based on morphological examination of flukes present in the Fasciola spp. population (n=4,725 adults) from ruminants

BL, body length; BW, body width.

Table 5 Molecular delineation of Fasciola species and types of hybridization among S-, M- and L-shaped liver flukes from ruminants and human samples in Vietnam based on the key nucleotide markers of ITS1 and ITS2 and mitochondrial nad1

ITS1, ITS2, internal transcribed spacer 1 and 2; Fhep, F. hepatica; F. gigantica; rDNA, ribosomal DNA; mtDNA, mitochondrial DNA; S, small; M, medium, L, large shape; Y, T or C; W, T or A.

299: F. hepatica possesses GTAC-sequence for a RsaI(+) at positions 296–299 in ITS1, while F. gigantica does not (GTAT/(RsaI(−));

327: At position 327 in ITS2, a nucleotide T (Thymine) present in F. hepatica and absent in F. gigantica [11,21,37].