Abstract: The present study examined sequence variations in the Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA) and two mitochondrial DNA (mtDNA) regions, namely cytochromec oxidase subunit 1 (cox1), NADH dehydrogenase subunits 1 (nad1), among Metorchis orientalis metacercaria isolates from Guangxi in China. The sequences of ITS, pcox1 and pnad1 were amplified from 6 individual M. orientalis metacercariae and sequenced. The relevant sequences of other 7 trematode species belonging to 6 genera in 4 families were downloaded from GenBank and their phylogenetic relationships were re-constructed using the combined pcox1 and pnad1 mt DNA sequences with Trichinella spiralis as outgroup. The results showed that sequences of ITS rDNA, pcox1 and pnad1 of M. orientalis were 1131, 654 and 650 bp, respectively and they were quite conserved among the M. orientalis isolates. However, they were quite different from that of other species, phylogenetic analysis of the combined pcox1 and pnad1 mt DNA sequences were able to distinguish M. orientalis from different species of the Opisthorchiidae and trematodes in other families. Therefore, the ITS, cox1 and nad1 mt DNA sequences provide effective genetic markers for the specific identification of trematodes of the Opisthorchiidae family and have implications for studying their population biology, genetic structure, as well as molecular epidemiology.

INTRODUCTION

Trematodes in the family Opisthorchiidae are divided into Opisthorchiina and Metoriinae which can infect mollusks and vertebrates. Some of the trematodes in Opisthorchiidae such as Clonorchis sinensis, Opisthorchis felineus and Metorchis orientalis are of zoonotic importance, infecting both humans and animals and causing death and health problem as well as significant economic losses (Lin et al., 2001; Shekhovtsov et al., 2009; Sohn, 2009). M. orientalis is recognized as one of the causative agents of trematode diseases in domestic animals and humans (Sohn et al., 1992; Cheng et al., 2005; Zhu et al., 2006). About 26 species of Metorchis have been reported worldwide of which 8 species parasitize in mammals and 18 species in birds. The final hosts of M. orientalis were always considered to be ducks or other poultry. However, Lin and Cheng (1986) firstly reported that cats, dogs were naturally infected by this trematode. It also can infect guinea pigs, rats, mice and domestic cats experimentally. Moreover, infection of humans with M. orientalis has been detected (Lin et al., 2001). The fish Psetidorasbora parvaas is the second intermediate host of this parasite in which the metacercaria stage develops (Cheng et al., 2005).

Currently, the identification and classification of M. orientalis is based on morphological characters, especially body length and width. However, it is difficult to accurately discriminate between M. orientalis and other Opisthorchiidae trematodes, especially at the metacercaria stage because of their morphological similarities (Yossepowitch et al., 2004; Schuster et al., 1999; Kang et al., 2008; Skov et al., 2008; Sherrard-Smith et al., 2009; Traub et al., 2009; Cai et al., 2010). The objectives of the present study were to determine the sequences of the Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA) and two mitochondrial DNA (mtDNA) regions namely cytochromec oxidase subunit 1 (cox1), NADH dehydrogenase subunits 1 (nad1) from M. orientalis metacercaria isolates from Guangxi in China and then to study the phylogenetic relationships among Opisthorchiidae trematodes using combined cox1 and nad1 sequences.

MATERIALS AND METHODS

Parasites and isolation of genomic DNA: M. orientalis isolates were collected from Guangxi Zhuang Nationality Autonomous Region, China. Sample codes, host and GenBank^TM accession number are shown in Table 1. The metacercariae were stored in 70% molecular grade ethanol and stored at -20°C before extraction of genomic DNA. Total genomic DNA was extracted from individual metacercariae by SDS/proteinase K treatment, column- purified (Wizard^® SV Genomic DNA Purification System, Promega) and eluted into 30 μL H₂O according to the manufacturer’s recommendations (Zhao et al., 2009a, b, 2010; Ai et al., 2010).

Enzymatic amplification and sequencing: The rDNA region comprising ITS-1, 5.8S and ITS-2 plus primer flanking sequences were amplified by Polymerase Chain Reaction (PCR) from trematode DNA using primers BD1 and BD2 (Luton et al., 1992). A portion of the cox1 gene (pcox1) was amplified with primers JB3 and JB4.5 (Bowles et al., 1992), part of the nad1 gene (pnad1) with primers nad1-F and nad1-R (Li et al., 2008a, b) (Table 2). PCR reactions (25 μL) were performed in 2 mM of MgCl₂ (2.5 mM for ITS and pnad1, 3 mM for pcox1), 2.5 μM of each primer, 2.5 μL 10x rTaq buffer, 0.2 mM of each dNTPs, 1.25 U of rTaq DNA polymerase (TAKARA) and 2 μL of DNA sample in a thermocycler (Biometra) under the following conditions: after an initial denaturation at 94°C for 5 min, then 94°C for 30 sec (denaturation); 50°C (for ITS and pnad1) or 55°C (for pcox1) for 30 sec (annealing); 72°C for 30 sec (extension) for 35 cycles, followed by a final extension at 72°C for 5 min.

These optimized cycling conditions for the specific and efficient amplification of individual ITS and mtDNA fragments were obtained after varying annealing temperatures.

Samples without genomic DNA (no-DNA controls) were included in each amplification run and in no case were amplicons detected in the no-DNA. Each amplicon (5 μL) was examined by agarose gel electrophoresis to validate amplification efficiency. Positive amplicons were selected, purified and sequenced using an ABI 377 automated DNA sequencer (using BigDye Terminator Chemistry) employing the same primers (individually) as used in the PCR.

Table 1:	Metacercaria samples of Metorchis orientalis from Guangxi, China used in the present study as well as their GenBankTM accession numbers for sequences of the Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA), a portion of mitochondrial DNA (mtDNA) cytochromec oxidase subunit 1 (pcox1) and NADH dehydrogenase subunits 1 (pnad1)

Table 2:	Sequences of primers used to amplify the Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA), a portion of cytochromec oxidase subunit 1 (pcox1) and NADH dehydrogenase subunits 1 (pnad1) of metacercaria samples of Metorchis orientalis from Guangxi, China

The ITS, pcox1 and pnad1 sequences are available from DDBJ, EMBL and GenBank™ under the accession numbers shown in Table 1.

Sequences analysis and reconstruction of phylogenetic relationships: The pcox1 and pnad1 sequences were separately aligned using the computer program Clustal x1.83 (Thompson et al., 1997). Sequence Differences (D) were calculated by pair-wise comparison using the formula D = 1-(M/L) in which M is the number of alignment positions at which the two sequences have a base in common and L is the total number of alignment positions over which the two sequences are compared (Chilton et al., 1995). To study the phylogenetic relationships between M. orientalis and other 7 trematode species belonging to 6 genera in 4 families, the combined pcox1 and pnad1 sequences of M. orientalis as well as that of Clonorchis sinensis, Opisthorchis felineus, Fasciola hepatica, Schistosoma japonicum, Schistosoma mansoni, Trichobilharzia regenti and Paragonimus westermani obtained from GenBank (Table 1) were used for phylogenetic analyses with Trichinella spiralis (NC_002681) as the outgroup (GenBank™ accession number can be shown in Table 1).

Three methods namely Neighbor Joining (NJ), Maximum Likelihood (ML) and Maximum Parsimony (MP) were used for phylogenetic re-constructions. NJ and MP analysis were carried out using PAUP 4.0 Beta 10 programme (Swofford, 2002) and ML analyses were performed using PUZZLE 4.1 (Strimmer and von Haeseler, 1996) under the default setting. The consensus tree was obtained after bootstrap analysis of 1000 replications and values above 50% were reported. Phylograms were drawn using the Tree View program version 1.65 (Page, 1996).

RESULTS AND DISCUSSION

Genomic DNA was prepared from 6 individual metacercariae from Guan gxi in China (Table 1). Amplicons of ITS, pcox1 and pnad1 (~1300, 720, 720 bp, respectively) were amplified individually and subjected to agarose gel electrophoresis. For each DNA region, no size variation was detected on agarose gel among any of the amplicons examined (Fig. 1).

Fig. 1:

Representative PCR products of metacercaria samples of Metorchis orientalis for the Internal Transcribed Spacers (ITS) of rDNA (upper), a portion of cytochrome c oxidase subunit 1 (pcox1, middle) and NADH dehydrogenase subunits 1 (pnad1, bottom) from Guangxi in China. Lanes 1-7 represent samples Momgx1, Momgx2, Momgx3, Momgx4, Momgx5, Momgx6 and negative control, respectively. M represents a DNA size marker (ordinate values in bp)

To examine sequence variations in the ITS and two mt DNA regions among isolates, the amplicons were subjected to direct sequencing. The sequences of ITS, pcox1 and pnad1 were 1131, 654 and 650 bp in length, respectively. The A+T contents of the sequences were 46.42-46.51% (ITS), 59.39-59.57% (pcox1) and 62.54-63.11% (pnad1), respectively. Sequence variations among M. orientalis isolates were 0.0-0.3% for ITS, 0.0-0.4% for pcox1 and 0.0-1.2% for pnad1. Sequence difference in the ITS and 5.8S between M. orientalis and Metorchis bilis (EU038154) were 4.0-4.2%, between M. orientalis and Opisthorchis felineus (EU038137) were 6.0-6.1% and between M. orientalis and Clonorchis sinensis (AF181892) were 10.0-10.1%.

Table 3:	Pairwise comparison of sequence differences (%) in the partial mitochondrial cytochrome c oxidase subunit 1 gene (pcox1, above the diagonal), NADH dehydrogenase subunits 1 genes (pnad1, below the diagonal) among Metorchis orientalis isolates from Guangxi in China

Fig. 2:

Phylogenetic relationship of Metorchis orientalis with other trematodes inferred by Neighbor-Joining (NJ), Maximum Parsimony (MP) and Maximum Likelihood (ML) analyses using the combined pcox1 and pnad1 sequences, with Trichinella spiralis as outgroup. Bootstrap values (in percentage) above 50% from 1,000 pseudo–replicates are shown for the NJ (the first value), MP (the second value) and ML analyses (the third value). weak = node resolved by method but very weak (<50%). Scale bar indicates an evolutionary distance of 10 substitutions per site in the sequence

Then, pcox1 and pnad1 mtDNA sequences were assessed whether they could provide a suitable marker for examining relationships between M. orientalis and other trematodes. In order to examine sequence differences in the pcox1 and pnad1 mtDNA among other trematodes, sequences of M. orientalis isolates in China were aligned into a consensus sequence. The genetic difference between Clonorchis and Opisthorchis in the combined pcox1 and pnad1 sequences was 22.7-23.5% for pcox1 and 24.1-26.5% for pnad1 and was 32.2-37.5% for pcox1 and 40.2-43.0% for pnad1 between Schistosoma and Trichobilharzia, respectively (Table 3).

The combined sequences of pcox1 and pnad1 mtDNA were aligned over a consensus length of 1304 bp. Topologies of the combined pcox1 and pnad1sequences inferred by different methods (NJ, MP and ML) with different building strategies and/or different distance models were similar (Fig. 2). The phylogenetic tree was consisted of two large clades: the first one contained all examined trematodes of the family Opisthorchiidae and the other one includes all other examined trematodes. Within the first clade, all the trematodes belonging to the family Opisthorchiidae were divided into two groups, C. sinensis and O. felineus. For the Opisthorchiidae cluster, M. orientalis isolates were grouped together and the isolates of C. sinensis and O. felineus were clustered together with high bootstrap value (>50%), respectively (Fig. 2). Within the second clade, Fasciola (Fasciolidae) trematodes and Paragonimus (Paragonimidae) flukes were clustered together, Schistosoma samples were clustered together, respectively. This clustering is in agreement with the results of traditional classifications.

CONCLUSION

The results of the present study were the first charactrization of M. orientalis metacercariae in China by a genetic approach using ITS rDNA, pcox1and pnad1 mtDNA as genetic markers. The combined pcox1 and pnad1 sequences are useful for re-construction of phylogenetic relationships between M. orientalis and other trematodes. The ITS, cox1 and nad1 mt DNA sequences provide effective genetic markers for the specific identification of trematodes of the Opisthorchiidae family and have implications for studying their population biology, genetic structure as well as molecular epidemiology.

ACKNOWLEDGEMENTS

Project supports were provided by grants from the Program for National S and T Major Program (Grant No. 2008ZX10004-011), National Key Technology R and D Program (Grant No.2008BAI56B03) and Special Technical Standards for Science and Technology Commission of Shanghai, China (Grant no 09DZ0503100).