Introduction
Dicrocoeliosis, caused by Dicrocoelium spp., is a worldwide parasitic disease of great economic significance and public health concern (González-Lanza et al., Reference González-Lanza, Manga-González and Del-Pozo-Carnero1993; Campo et al., Reference Campo, Manga-González and González-Lanza2000; Jeandron et al., Reference Jeandron, Rinaldi, Abdyldaieva, Usubalieva, Steinmann, Cringoli and Utzinger2011). Dicrocoelium dendriticum, one of the common causative agents of dicrocoeliosis in small ruminants, mainly inhabits the bile ducts and gall bladder of hosts (Otranto & Traversa, Reference Otranto and Traversa2003; Taira et al., Reference Taira, Shirasaka, Taira, Ando and Adachi2006; Maurelli et al., Reference Maurelli, Rinaldi, Capuano, Perugini, Veneziano and Cringoli2007). In general, economic losses caused by dicrocoeliosis are difficult to quantify, in terms of concomitant gastrointestinal and lung infections caused by nematodes and distomatids (Otranto & Traversa, Reference Otranto and Traversa2003). However, severe infections may lead to severe economic losses, due to direct losses caused by liver condemnation, and indirect ones such as low meat and milk production, costs incurred by use of anthelminthics, and even death of lambs and calves (Manga-González et al., Reference Manga-González, González-Lanza, Cabanas and Campo2001; Otranto & Traversa, Reference Otranto and Traversa2002; Manga-González & González-Lanza, Reference Manga-González and González-Lanza2005).
To control dicrocoeliosis effectively, an accurate characterization of D. dendriticum at different taxonomic levels is essential. However, traditional characterizations of D. dendriticum, mainly based on detecting eggs in the faeces of live animals and finding adults in the liver during necropsy, based on morphological characters (Campo et al., Reference Campo, Manga-González and González-Lanza2000; Manga-González & González-Lanza, Reference Manga-González and González-Lanza2005), could not differentiate between closely related species and reveal intra-species genetic diversity, which enhanced the difficulty of controlling dicrocoeliosis (Rollinson et al., Reference Rollinson, Walker and Simpson1986; Sandoval et al., Reference Sandoval, Manga-González, Campo, García, Castro and Pérez de la Vega1999). Recently, the DNA-based technique provided an alternative approach for identification and population genetic study of parasites (Gasser et al., Reference Gasser, Rossi and Zhu1999, Reference Gasser, Bott, Chilton, Hunt and Beveridge2008; Chitimia et al., Reference Chitimia, Lin, Cosoroaba, Braila, Song and Zhu2009; Martínez-Ibeas et al., Reference Martínez-Ibeas, Martínez-Valladares, González-Lanza, Miñambres and Manga-González2011; Huang et al., Reference Huang, Zhao, Fu, Xu, Wang, Wu, Zou and Zhu2012; Wang et al., Reference Wang, Gao, Zhu and Zhao2012). Nuclear ribosomal DNA (rDNA) has been used as a suitable marker for species characterization of nematodes, trematodes and cestodes (Huang et al., Reference Huang, He, Wang and Zhu2004; Zhu et al., Reference Zhu, Podolska, Liu, Yu, Chen, Lin, Luo, Song and Lin2007; Orosová et al., Reference Orosová, Ivica, Eva and Marta2010). The 18S and internal transcribed spacer (ITS)-2 rDNA could clearly differentiate between D. dendriticum and D. chinensis (Otranto et al., Reference Otranto, Rehbein, Weigl, Cantacessi, Parisi, Lia and Olson2007). In addition, the 28S and ITS-2 rDNA have been used to identify D. dendriticum and D. hospes (Maurelli et al., Reference Maurelli, Rinaldi, Capuano, Perugini, Veneziano and Cringoli2007). Recently, larval stages of D. dendriticum in molluscan and ant intermediate hosts were detected using a polymerase chain reaction (PCR) technique based on mitochondrial cox1 and ITS-2 rDNA sequences (Martínez-Ibeas et al., Reference Martínez-Ibeas, Martínez-Valladares, González-Lanza, Miñambres and Manga-González2011). However, prior to the present study, no information had been provided on genetic variations of D. dendriticum in different hosts and geographical origins. Here, we examined sequence variability in ITS rDNA, among D. dendriticum isolates from different geographical origins and hosts in Shaanxi province, north-western China, and also analysed phylogenetic relationships of these D. dendriticum isolates based on sequences of ITS-2 rDNA.
Materials and methods
Isolation of genomic DNA
Up to 25 adult worms of D. dendriticum samples were isolated from four naturally infected sheep and 12 goats from four geographical regions in Shaanxi province, north-western China. The geographic distances between each two sampling sites ranged from 20 km (between Yangling district and Jingyang county) to 700 km (between Jingyang county and Shenmu county). Sample codes, hosts and GenBank accession numbers are listed in table 1. All parasites were identified to species according to morphological features (Taira et al., Reference Taira, Shirasaka, Taira, Ando and Adachi2006; Otranto et al., Reference Otranto, Rehbein, Weigl, Cantacessi, Parisi, Lia and Olson2007) and ITS-2 rDNA sequences published previously (Maurelli et al., Reference Maurelli, Rinaldi, Capuano, Perugini, Veneziano and Cringoli2007; Otranto et al., Reference Otranto, Rehbein, Weigl, Cantacessi, Parisi, Lia and Olson2007), then washed with physiological saline and fixed in 70% ethanol. Genomic DNA (gDNA) of each worm was extracted using spin column purification (EZNA Genomic DNA Purification System, OMEGA, Georgia, USA) according to the manufacturer's instructions.
Table 1 The geographical origins and hosts of Dicrocoelium dendriticum and other species in this genus together with their GenBank accession numbers for sequences of the internal transcribed spacer of nuclear ribosomal DNA, and all sample codes referring to D. dendriticum.
a Otranto et al. (Reference Otranto, Rehbein, Weigl, Cantacessi, Parisi, Lia and Olson2007).
b Maurelli et al. (Reference Maurelli, Rinaldi, Capuano, Perugini, Veneziano and Cringoli2007).
Amplification of the ITS-1, 5.8S and ITS-2 rDNA
The rDNA regions containing complete ITS-1, 5.8S and ITS-2 rDNA plus partial sequences of 18S and 28S rDNA were amplified by polymerase chain reaction (PCR) from each D. dendriticum DNA sample, using the universal primers BD1 and BD2 described previously (Luton et al., Reference Luton, Walker and Blair1992; Ali et al., Reference Ali, Ai, Song, Ali, Lin, Seyni, Issa and Zhu2008). Negative controls, including samples without gDNA and host DNA (sheep and goat), were also included in each PCR amplification. The amplification efficiency was validated by agarose gel (1%) electrophoresis with 5 μl of each PCR product.
Cloning, sequencing, genetic analysis and phylogenetic reconstruction
Positive PCR amplicons were purified using spin columns (DNA-Preps Purification System, Sangon, China). Purified products were ligated with pMD19-T plasmid vector (Takara, Dalian, China) according to the manufacturer's recommendations, and transformed into Escherichia coli DH5α-competent cells (Takara). The transformants were identified by PCR amplification, with positive transformants sent to Songon Company (Shanghai, China) for sequencing in both directions.
The 5′ and 3′ ends of the ITS-1, 5.8S and ITS-2 rDNA fragments of each D. dendriticum isolate were determined by aligning with available sequences of D. dendriticum (GenBank accession number HM358027) and Euparyphium albuferensis (AJ564384). Each rDNA fragment from different isolates was aligned separately using Clustal X 1.83 (Thompson et al., Reference Thompson, Gibson, Plewniak, Jeanmougin and Higgins1997).
Since there were only two mutation sites in 5.8S rDNA sequences within D. dendriticum, sequences of ITS-1 and ITS-2 rDNA were used for further study. The genetic variations (D) between D. dendriticum individuals were determined using the formula D= 1 − (M/L) described by Chilton et al. (Reference Chilton, Gasser and Beveridge1995). The inter-species genetic differences among species in genus Dicrocoelium were evaluated using the Megalign procedure in DNASTAR 5.0 (Burland, Reference Burland2000). In addition, Mega 4.0 software (Tamura et al., Reference Tamura, Dudley, Nei and Kumar2007) was used to calculate base composition, transitions and transversions.
There were no available ITS-1 rDNA sequences for Dicrocoelium species, so only the ITS-2 rDNA was used to infer phylogenetic relationships of Dicrocoelium spp. (table 1). After ambiguous regions were excluded, using Gblocks online server (http://molevol.cmima.csic.es/castresana/Gblocks_server.html), the remaining sequences were aligned and used for phylogenetic analyses using the neighbour-joining (NJ) methods in MEGA 4.0 (Tamura et al., Reference Tamura, Dudley, Nei and Kumar2007), with the substitution model of the Kimura 2-parameter method. The consensus tree was obtained after bootstrap analysis with 1000 replications, with values above 50% reported. The Tree View program version 1.65 (Page, Reference Page1996) was used to draw phylograms.
Results and discussion
Amplicons containing complete ITS rDNA regions, approximately 1200 bp in length, of 25 D. dendriticum gDNA samples from four geographical regions in Shaanxi province were amplified individually, with the same size detected by agarose gel electrophoresis (data not shown).
Intra-species variations within D. dendriticum isolates and inter-species differences among species within genus Dicrocoelium in ITS rDNA regions were examined. The lengths of ITS rDNA for D. dendriticum were 1144 bp, with 749 bp, 161 bp and 234 bp for ITS-1, 5.8S and ITS-2, respectively. The AT contents of ITS-1 and ITS-2 rDNA sequences for D. dendriticum samples in this present study were 47.26–47.53% and 51.28–52.56%, respectively. No sequence difference in ITS-1 rDNA was detected for samples from Jingyang county, but the genetic variations in ITS-1 rDNA were 0–0.1%, 0–0.4% and 0–0.3% for samples from Yangling district, Shenmu county and Zhouzhi county, respectively. For ITS-2 rDNA, the genetic variations were 0–0.4% for samples from both Jingyang county and Yangling district, 0–1.3% for Shenmu county and 0–0.9% for Zhouzhi county. Among D. dendriticum isolates from goats in Shaanxi province, the intra-species genetic variations in ITS-1 and ITS-2 rDNA were 0–0.5% and 0–1.3%, respectively. For samples from sheep in this province, sequence variations in ITS-2 rDNA were 0–0.4%, whereas no genetic variation was detected in ITS-1 rDNA. For all D. dendriticum samples in Shaanxi province, the intra-species sequence variations in ITS-1 rDNA were 0–0.5%, including three C ↔ T transitions and four transversions (A ↔ C, n= 1; G ↔ C, n= 2 and/or G ↔ T, n= 1), and 0–1.3% in the ITS-2 rDNA, including four transitions (C ↔ T, n= 2 and/or A ↔ G, n= 2).
The genetic variations of D. dendriticum isolates from different countries and hosts were determined using the ITS-2 rDNA sequences available in GenBank. The sequence differences in ITS-2 rDNA were 0–0.9%, 0–1.3% and 0–1.3% between D. dendriticum samples from Shaanxi province of China and isolates from Iran, Slovakia and Japan, respectively. The genetic variations were 0–1.3% among samples from sheep, goats, cattle and Japanese serow. Moreover, the intra-species variations were 0–1.3% between samples from sheep and goats, 0.4–0.9% between sheep and cattle, 0.4–0.9% between sheep and Japanese serow, 0.4–1.3% between goats and cattle, and 0.4–1.3% between goats and Japanese serow. No variation was detected for samples from cattle and Japanese serow. However, high inter-species sequence differences were detected among members of Dicrocoelium species (table 1), with 3.4–12.3% variation in ITS-2 rDNA.
To further determine the phylogenetic position of D. dendriticum isolates in Shaanxi province, the NJ phylogenetic tree among Dicrocoelium spp. was reconstructed based on the sequences of ITS-2 rDNA (fig. 1). All D. dendriticum isolates in the present study were grouped with reference D. dendriticum isolates from sheep and goat, and D. dendriticum isolates from cattle and Japanese serow were clustered in the sister clade. In addition, D. orientalis was located in a clade of D. chinensis isolates with high bootstrap value, suggesting that the two Dicrocoelium species are one species. However, D. dendriticum samples isolated from different origins or hosts were grouped in one clade, and isolates from the same region or host were located in different clades. These results indicated that the ITS-2 rDNA was not a suitable marker to infer phylogenetic relationships of D. dendriticum samples from different regions and hosts, but may be a useful marker to study phylogenetic relationships of Dicrocoelium species.
Fig. 1 Phylogenetic analysis of Dicrocoelium spp. based on ITS-2 rDNA sequences using the neighbour–joining (NJ) method; the consensus tree was obtained after bootstrap analysis with 1000 replications, with values above 50% reported.
In conclusion, the ITS rDNA of D. dendriticum in small ruminants from Shaanxi province was characterized, and the genetic variations in ITS-2 rDNA of D. dendriticum isolated from different geographical origins and hosts were also determined. The ITS-2 rDNA provided a suitable marker to infer phylogenetic relationships of Dicrocoelium species. These findings provided basic information for further study of molecular epidemiology and control of D. dendriticum infection in this province as well as in the world.
Acknowledgements
The authors alone are responsible for doing the research and writing this paper.
Financial support
This work was supported in part by grants from the Basic Research Key Program (grant number ZD2012010); and the Fund for Basic Scientific Research Program in Northwest A&F University, PR China (grant number QN2012018).
Statement of interest
None.
