Abstract: In the present study, a portion of the 18S and 28S ribosomal DNA (rDNA) sequences of 35 Schistosoma japonicum isolates representing three geographical strains from mainland China, the Philippines and Japan were amplified and compared and phylogenetic relationships were also reconstructed by Unweighted Pair-Group Method with Arithmetic averages (UPGMA) using combined 18S and 28S rDNA sequences as well as the corresponding sequences of other species belonging to the Schistosoma genus available in the public database. The results indicated that the partial 18S and 28S rDNA sequences of all S. japonicum isolates were 745 and 618 bp, respectively and displayed low genetic variation among S. japonicum strains and isolates. Phylogenetic analysis revealed that the combined 18S and 28S rDNA sequences were not able to distinguish S. japonicum isolates from three geographical origins but provided an effective molecular marker for the inter-species phylogenetic analysis and differential identification of different Schistosoma species.

INTRODUCTION

Schistosomiasis caused by trematodes of the genus Schistosoma is a parasitic disease of medical and veterinary importance in a number of countries with an estimated approximately 200 million people being infected and approximately 200,000 deaths per year (Chitsulo et al., 2004; Taylor, 2008; Huyse et al., 2009). Among six human schistosomes (Schistosoma japonicum, Schistosoma haematobium, Schistosoma mansoni (Schistosoma intercalatum = S. guineensis), Schistosoma mekongi and Schistosoma malayensis), three main species (S. japonicum, S. haematobium and S. mansoni) are reported to have significant human health and social-economic impact (Zhao et al., 2009a). Schistosomiasis japonica, one of the most serious schistosomiasis caused by S. japonicum was mainly distributed in China, Japan, the Philippines and parts of Indonesia and caused various degrees of morbidity and mortality (Zhou et al., 2005; Zhao et al., 2009a, b). In addition to the six human schistosomes, other 13 Schistosoma species have been identified as parasites in animals. S. hippopotami and S. edwardiense are found in hippopotamus (Morgan et al., 2003), S. mattheei, S. bovis, S. leiperi, S. margrebowiei, S. curassoni, S. indicum, S. spindale and S. nasale found in ruminant animals such as cattle, buffalo, sheep, goat and lechwe (De Bont et al., 1994; Vercruysse et al., 2003; Singh et al., 2004; Vercruysse and Gabriel, 2005; Littlewood et al., 2006; Sato et al., 2008) and S. rodhaini, S. incognitum, S. sinensium mainly inhabited in rodents and carnivores (Bunnag et al., 1983; Walker et al., 1989; Zhang et al., 2001).

Ribosomal DNA (rDNA) forms a tandem array of repeat sequences and each repeat includes 18S, 5.8S and 28S subunits separated by spacers (Zhao et al., 2011). The 18S and 28S rDNA sequences have been identified as good genetic markers for identification and phylogenetic studies of members of the genus Schistosoma (Johnston et al., 1993; Barker and Blair, 1996; Attwood et al., 2002; Lockyer et al., 2003; Webster et al., 2006). Recent studies also have demonstrated that different S. japonicum strains from mainland China and the Philippines has extensive homology in 18S rDNA sequences and 28S rDNA-D2 domain was also relatively stable among species (Yu et al., 2000; Li et al., 2008). But sequence variation in 18 and 28S rDNA among S. japonicum strains from mainland China, the Philippines and Japan have not been investigated comprehensively.

The objectives of the present study were to examine sequence variation in the 18S and 28S rDNA among S. japonicum isolates from different endemic regions in mainland China, the Philippines and Japan and to reconstruct the phylogenetic relationships among members of the Schistosoma genus using the combined partial 18S and 28S rDNA sequences.

MATERIALS AND METHODS

Parasites and isolation of genomic DNA: The 35 S. japonicum isolates were collected from the endemic areas in mainland China, the Philippines and Japan with sample codes, geographical origin and gender shown in Table 1. The male and female adult parasites were fixed in 70% molecular grade ethanol and stored at -20°C before extraction of genomic DNA. Total genomic DNA was extracted from individual parasites by SDS/proteinase K treatment, column-purified (Wizard^® SV Genomic DNA Purification System, Promega) and eluted into 60 μL H₂O according to the manufacturer’s recommendations.

Enzymatic amplification and sequencing: A portion of the 18S (p18S) and 28S rDNA were amplified with primers 18Su and 18Sd, 28Su and 28Sd, respectively (Table 2). PCR reactions (25 μL ) were performed in 2 mM of MgCl₂, 2.5 μM of each primer, 2.5 μL 10xrTaq buffer, 0.2 mM of each dNTPs, 1.25 U of rTaq DNA polymerase (Takara) and 1 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 1 min (denaturation); 45°C for 30 sec (annealing); 72°C for 1 min (extension) for 35 cycles followed by a final extension at 72°C for 10 min. These optimized cycling conditions for the specific and efficient amplification of both rDNA fragments were obtained after adjusting annealing temperatures. Each amplicon (4 μL) was examined by agarose (1%) gel electrophoresis to validate amplification efficiency. The p18S and p28S amplicons of 35 samples were sequenced by BGI-Guangzhou company from both directions using the same primers as used in primary amplification.

Sequences alignment and analysis: Sequences of the p18S and p28S rDNA were separately aligned using the computer program Clustal X 1.81 (Thompson et al., 1997). Meanwhile, Megalign procedure within the DNAStar 5.0 (Burland, 2000) was also used to analyze sequence similarity and to calculate transition and transversion.

Table 1:	Information of Schistosoma samples used in the present study

Table 2:	Sequences of primers used to amplify a portion of the 18S and 28S ribosomal DNA from Schistosoma japonicum isolates from mainland China, the Philippines and Japan

These sequences were used for phylogenetic analyses. The sequences of the two rRNA genes were concatenated into single alignments. The Unweighted Pair-Group Method based on Arithmetic averages (UPGMA) (Sneath and Sokal, 1973) in MEGA v. 4.0 (Tamura et al., 2007) was carried out to examine the genetic relationship, starting from a distance matrix based on the Kimura 2-parameter index (Kimura, 1980). The consensus tree was obtained after bootstrap analysis with 1, 000 replications. The pairwise comparisons were made of the level of sequence differences (Chilton et al., 1995). To study the phylogenetic relationships of Schistosoma, the corresponding sequences of other Schistosoma species/isolates namely; S. intercalatum, S. rodhaini, S. spindale, S. bovis, S. edwardiense, S. haematobium, S. hippopotam, S. incognitum, S. indicum, S. japonicum, S. leiperi, S. malayensis, S. mattheei, S. mekongi, S. nasale, S. sinensium and S. mansoni obtained from GenBank were also used for phylogenetic analyses. The phylograms were drawn using the Tree View program version 1.65 (Page, 1996).

RESULTS AND DISCUSSION

Genomic DNA was prepared from 35 individual adult trematodes (including male and female S. japonicum) representing three geographical strains from mainland China, the Philippines and Japan. Amplicons of p18S and p28S rDNA (~750 and 620 bp, respectively) were amplified. For each rDNA fragment, no size variation was detected on 1% agarose gel among any of the amplicons examined in dicating that the PCR primers and reactions of this study had good specificity (Fig. 1).

To examine sequence variations in the two rDNA fragments among three geographical strains, amplicons of p18S and p28S rDNA were subjected to direct sequencing. The sequences of p18S and p28S rDNA were 745 and 618 bp in length, respectively. One variable nucleotide position was identified in the sequences of p18S and three in p28S rDNA with intra-specific variation of 0.14% (1/704) and 0.51% (3/586) for p18S and p28S rDNA, respectively.

Fig. 1:

Representative PCR products for a portion of the 18S (upper) and 28S (bottom) ribosomal DNA of Schistosoma japonicum isolates in mainland China, the Philippines and Japan. Lanes 1-18 represent samples SjYeM55, SjHyM54, SjZjF60, SjLeM1, SjLeM4, SjLeF1, SjMiM1, SjMiF1, SjMiF4, SjSoM3, SjSoM4, SjSoF2, SjAsM1, SjAsM2, SjAsF3, SjYYM2, SjYYM3, SjYYF1, respectively (cf. Table 1). Lane 19 represents no-DNA control. M represents a DNA size marker (ordinate values in bp)

Fig. 2:	Phylogenetic relationships of Schistosoma species/isolates inferred by UPGMA analysis using the combined sequences of partial 18S and p28S ribosomal DNA. Numbers at nodes indicate bootstrap values (%) resulting from UPGMA tree

There were one transitions (A<->G) with intra-specific variation for p18S and two (A<->G) and one (C<->T) transitions for p28S rDNA. It appeared that the main transition was A<->G and there was no transversion within these two fragments. The low variations between the p18S and p28S rDNA were consistent with previous reports (Yu et al., 2000; Li et al., 2008).

The combined sequences of p18S and p28S rDNA were aligned over a consensus length of 1,290 bp. The phylogenetic relationships among the individual S. japonicum isolates were constructed by UPGMA analyses (Fig. 2). The phylogenetic tree consisted of two large clades.

All of the S. japonicum isolates from mainland China, the Philippines and Japan clustered in the S. japonicum clade, grouped with S. malayensis and S. mekongi and sistered to S. sinensium. All of the other Schistosoma species grouped in the other large clade with high bootstrap values.

The combined sequences of p18S and p28S rDNA allowed the unequivocal differentiation of all the Schistosoma species examined in the present study which is consistent with previous studies (Johnston et al., 1993; Littlewood and Johnston, 1995; Attwood et al., 2002; Morgan et al., 2003; Webster et al., 2006).

CONCLUSION

The present study revealed low level variation in p18S and p28S rDNA sequences among 35 S. japonicum isolates from mainland China, the Philippines and Japan. The combined p18S and p28S rDNA sequences were highly conserved and could not distinguish S. japonicum isolates from three geographical origins by phylogenetic analysis but could differentiate species in the Schistosoma genus suggesting that the p18S and p28S rDNA sequences were not suitable markers for studying population relationships among S. japonicum isolates but is an effective genetic marker for inter-species phylogenetic analysis of Schistosoma and identification of schistosomes.

ACKNOWLEDGEMENTS

Project support was provided in part by the National Basic Research Program (973 program) of China (Grant No. 2007CB513104), the special funds for talents in Northwest A and F University, the National Natural Science Foundation of China (Grant No. 30960280), the Yunnan Provincial program for Introducing High-level Scientists (Grant No. 2009CI125) and the program for Changjiang scholars and innovative research team in university (Grant No. IRT0723). Professor Baozhen Qian of Bioengineering Institute, Zhejiang Academy of Medical Sciences, China was thanked for providing some S. japonicum samples used in the present study.