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Characterization of Dicrocoelium dendriticum isolates from small ruminants in Shaanxi Province, north-western China, using internal transcribed spacers of nuclear ribosomal DNA

Published online by Cambridge University Press:  26 July 2013

Q.Q. Bian,
G.H. Zhao,
Y.Q. Jia,
Y.Q. Fang,
W.Y. Cheng,
S.Z. Du,
X.T. Ma and
Q. Lin
Q.Q. Bian
Affiliation:
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi Province712100, PR China
G.H. Zhao*
Affiliation:
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi Province712100, PR China
Y.Q. Jia
Affiliation:
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi Province712100, PR China
Y.Q. Fang
Affiliation:
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi Province712100, PR China
W.Y. Cheng
Affiliation:
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi Province712100, PR China
S.Z. Du
Affiliation:
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi Province712100, PR China
X.T. Ma
Affiliation:
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi Province712100, PR China
Q. Lin*
Affiliation:
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi Province712100, PR China
*
*Fax: +86-29-87081762 E-mails: zgh083@163.com (G.H. Zhao); yllinqing@126.com (Q. Lin)
*Fax: +86-29-87081762 E-mails: zgh083@163.com (G.H. Zhao); yllinqing@126.com (Q. Lin)
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Abstract

The genetic variations in internal transcribed spacers (ITS) spanning ITS-1, 5.8S and ITS-2 rDNA of Dicrocoelium dendriticum, isolated from sheep and goats in four geographical regions in Shaanxi province, were examined. The lengths of ITS-1, 5.8S and ITS-2 rDNA sequences for D. dendriticum were 749 bp, 161 bp and 234 bp, respectively. Intra-specific sequence variations of D. dendriticum were 0–0.5% for ITS-1 and 0–1.3% for ITS-2 rDNA, while the inter-specific variations among species in genus Dicrocoelium in ITS-2 rDNA were 3.4–12.3%. Phylogenetic analysis based on sequences of ITS-2 rDNA showed that all D. dendriticum isolates in the present study were grouped with reference D. dendriticum isolates from sheep and goats, and D. dendriticum isolates from cattle and Japanese serow were clustered in a sister clade. However, the phylogenetic tree could not reveal geographically genetic relationships of D. dendriticum isolates in different origins and hosts. These findings provided basic information for further study of molecular epidemiology and control of D. dendriticum infection in Shaanxi province as well as in the world.

Information

Type
Short Communications
Information
Journal of Helminthology , Volume 89 , Issue 1 , January 2015, pp. 124 - 129
Copyright
Copyright © Cambridge University Press 2013 

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.

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.

References

Ali, H., Ai, L., Song, H.Q., Ali, S., Lin, R.Q., Seyni, B., Issa, G. & Zhu, X.Q. (2008) Genetic characterisation of Fasciola samples from different host species and geographical localities revealed the existence of F. hepatica and F. gigantica in Niger. Parasitology Research 102, 1021–1024.Google Scholar
Burland, T.G. (2000) DNASTAR's Lasergene sequence analysis software. Methods in Molecular Biology 132, 71–91.Google Scholar
Campo, R., Manga-González, M.Y. & González-Lanza, C. (2000) Relationship between egg output and parasitic burden in lambs experimentally infected with different doses of Dicrocoelium dendriticum (Digenea). Veterinary Parasitology 87, 139–149.CrossRefGoogle ScholarPubMed
Chilton, N.B., Gasser, R.B. & Beveridge, I. (1995) Differences in a ribosomal DNA sequence of morphologically indistinguishable species within the Hypodontus macropi complex (Nematoda: Strongyloidea). International Journal for Parasitology 25, 647–651.Google Scholar
Chitimia, L., Lin, R.Q., Cosoroaba, I., Braila, P., Song, H.Q. & Zhu, X.Q. (2009) Molecular characterization of hard ticks from Romania by sequences of the internal transcribed spacers of ribosomal DNA. Parasitology Research 105, 1479–1482.CrossRefGoogle ScholarPubMed
Gasser, R.B., Rossi, L. & Zhu, X.Q. (1999) Identification of Nematodirus species (Nematoda: Molineidae) from wild ruminants in Italy using ribosomal DNA markers. International Journal for Parasitology 29, 1809–1817.Google Scholar
Gasser, R.B., Bott, N.J., Chilton, N.B., Hunt, P. & Beveridge, I. (2008) Toward practical, DNA-based diagnostic methods for parasitic nematodes of livestock – bionomic and biotechnological implications. Biotechnology Advances 26, 325–334.CrossRefGoogle ScholarPubMed
González-Lanza, C., Manga-González, M.Y. & Del-Pozo-Carnero, P. (1993) Coprological study of the Dicrocoelium dendriticum (Digenea) egg elimination by cattle in highland areas in León Province, northwest Spain. Parasitology Research 79, 488–491.Google Scholar
Huang, W.Y., He, B., Wang, C.R. & Zhu, X.Q. (2004) Characterisation of Fasciola species from Mainland China by ITS-2 ribosomal DNA sequence. Veterinary Parasitology 120, 75–83.Google Scholar
Huang, S.Y., Zhao, G.H., Fu, B.Q., Xu, M.J., Wang, C.R., Wu, S.M., Zou, F.C. & Zhu, X.Q. (2012) Genomics and molecular genetics of Clonorchis sinensis: current status and perspectives. Parasitology International 61, 71–76.Google Scholar
Jeandron, A., Rinaldi, L., Abdyldaieva, G., Usubalieva, J., Steinmann, P., Cringoli, G. & Utzinger, J. (2011) Human infections with Dicrocoelium dendriticum in Kyrgyzstan: the tip of the iceberg? Journal of Parasitology 97, 1170–1172.Google Scholar
Luton, K., Walker, D. & Blair, D. (1992) Comparisons of ribosomal internal transcribed spacers from two congeneric species of flukes (Platyhelminthes: Trematoda: Digenea). Molecular and Biochemical Parasitology 56, 323–327.Google Scholar
Manga-González, M.Y. & González-Lanza, C. (2005) Field and experimental studies on Dicrocoelium dendriticum and dicrocoeliosis in northern Spain. Journal of Heminthology 79, 291–302.Google Scholar
Manga-González, M.Y., González-Lanza, C., Cabanas, E. & Campo, R. (2001) Contributions to and review of dicrocoeliosis, with special reference to the intermediate hosts of Dicrocoelium dendriticum. Parasitology 123, 91–114.Google Scholar
Martínez-Ibeas, A.M., Martínez-Valladares, M., González-Lanza, C., Miñambres, B. & Manga-González, M.Y. (2011) Detection of Dicrocoelium dendriticum larval stages in mollusk and ant intermediate hosts by PCR, using mitochondrial and ribosomal internal transcribed spacer (ITS-2) sequences. Parasitology 138, 1916–1923.Google Scholar
Maurelli, M.P., Rinaldi, L., Capuano, F., Perugini, A.G., Veneziano, V. & Cringoli, G. (2007) Characterization of the 28S and the second internal transcribed spacer of ribosomal DNA of Dicrocoelium dendriticum and Dicrocoelium hospes. Parasitology Research 101, 1251–1255.Google Scholar
Orosová, M., Ivica, K.H., Eva, B. & Marta, S. (2010) Karyotype, chromosomal characteristics of multiple rDNA clusters and intragenomic variability of ribosomal ITS2 in Caryophyllaeides fennica (Cestoda). Parasitology International 59, 351–357.CrossRefGoogle ScholarPubMed
Otranto, D. & Traversa, D. (2002) A review of dicrocoeliosis of ruminants including recent advances in the diagnosis and treatment. Veterinary Parasitology 107, 317–335.Google Scholar
Otranto, D. & Traversa, D. (2003) Dicrocoeliosis of ruminants: a little known fluke disease. Trends in Parasitology 19, 12–15.Google Scholar
Otranto, D., Rehbein, S., Weigl, S., Cantacessi, C., Parisi, A., Lia, R.P. & Olson, P.D. (2007) Morphological and molecular differentiation between Dicrocoelium dendriticum (Rudolphi, 1819) and Dicrocoelium chinensis (Sudarikov and Ryjikov, 1951) Tang and Tang, 1978 (Platyhelminthes: Digenea). Acta Tropica 104, 91–98.Google Scholar
Page, R.D. (1996) TreeView: an application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences 12, 357–358.Google Scholar
Rollinson, D., Walker, T.K. & Simpson, A.J. (1986) The application of recombinant DNA technology to problems of helminth identification. Parasitology 92, 53–71.Google Scholar
Sandoval, H., Manga-González, Y., Campo, R., García, P., Castro, J.M. & Pérez de la Vega, M. (1999) Preliminary study on genetic variability of Dicrocoelium dendriticum determined by random amplified polymorphic DNA. Parasitology International 48, 21–26.Google Scholar
Taira, K., Shirasaka, S., Taira, N., Ando, Y. & Adachi, Y. (2006) Morphometry of lancet flukes found in Japanese sika deer (Cervus nippon centralis) captured in Iwate Prefecture, Japan. Journal of Veterinary Medical Science 68, 375–377.Google Scholar
Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24, 1596–1599.Google Scholar
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. & Higgins, D.G. (1997) The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25, 4876–4882.Google Scholar
Wang, C.R., Gao, J.F., Zhu, X.Q. & Zhao, Q. (2012) Characterization of Bunostomum trigonocephalum and Bunostomum phlebotomum from sheep and cattle by internal transcribed spacers of nuclear ribosomal DNA. Research in Veterinary Science 92, 99–102.Google Scholar
Zhu, X.Q., Podolska, M., Liu, J.S., Yu, H.Q., Chen, H.H., Lin, Z.X., Luo, C.B., Song, H.Q. & Lin, R.Q. (2007) Identification of anisakid nematodes with zoonotic potential from Europe and China by single-strand conformation polymorphism analysis of nuclear ribosomal DNA. Parasitology Research 101, 1703–1707.CrossRefGoogle ScholarPubMed
Figure 0

Table 1The 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.

Figure 1

Fig. 1Phylogenetic 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.