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Veterinary Parasitology 153 (2008) 379–383 www.elsevier.com/locate/vetpar
Characterization of rDNA sequences from Syphacia obvelata, Syphacia muris, and Aspiculuris tetraptera and development of a PCR-based method for identification Joan Dee C. Parel, Jedhan U. Galula, Hong-Kean Ooi * Department of Veterinary Medicine, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung, Taiwan Received 3 November 2007; received in revised form 28 January 2008; accepted 1 February 2008
Abstract To differentiate the morphologically similar pinworms of the common laboratory rodents, such as Syphacia obvelata and Syphacia muris, we amplified and sequenced the region spanning the internal transcribed spacer 1 (ITS-1), 5.8S gene, and ITS-2 of the ribosomal DNA followed by designing of species-specific primers for future use in the identification of the worms. It was observed that S. obvelata, S. muris and Aspiculuris tetraptera can be differentiated from each other based on their rDNA sequences. This is the first report of the ITS-1, 5.8S, and ITS-2 of the rDNA of the three aforementioned rodent pinworm species. The use of restriction endonucleases, AluI or RsaI, further allowed the delineation of the three species. Moreover, we also constructed speciesspecific primers that were designed for unique regions of the ITS-2 of the three species. This approach allowed their specific identification with no amplicons being amplified from heterogenous DNA samples, and sequencing confirmed the identity of the sequences amplified. Thus, the use of these specific primers along with PCR-RFLP can serve as useful tools for the identification of pinworms in rats, mice, and wild rodents. # 2008 Elsevier B.V. All rights reserved. Keywords: Pinworms; Syphacia obvelata; Syphacia muris; Aspiculuris tetraptera; ITS-2; rDNA
1. Introduction Pinworms of the Order Oxyurina (Syphacia obvelata, Syphacia muris and Aspiculuris tetraptera) are important parasites of laboratory rats and mice, commonly infecting these animals with high prevalence even in well-managed colonies (Baker, 1998; Bazzano et al., 2002; Perec-Matysiak et al., 2006). Effects of pinworms on research include induction of proliferation of T and B lymphocytes in spleen and lymph nodes and occasional germinal center formation (Beattie et al.,
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(H.-K. Ooi). 0304-4017/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.02.001
1980), reduction of the occurrence of adjuvant-induced arthritis (Pearson and Taylor, 1975) and decreased intestinal transport of water and electrolytes in rats due to pinworm infection (Lu¨bcke et al., 1992). It was also shown that pinworms alter the growth (Wagner, 1988), hematopoiesis (Bugarski et al., 2006) and the humoral response to nonparasitic stimuli (Sato et al., 1995) of mice. Pinworms also modulate the immune system of mice by inducing a Th2-biased immune response (Michels et al., 2006). Identification of the parasite is deemed important for taxonomy, diagnosis and whenever their effects are being studied. Currently, identification of Syphacia spp. infecting the rat or mice is done by examining the male worms for the location of the mamelons (Ooi et al., 1994; Bazzano et al., 2002; Pinto et al., 2001). However,
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male worms are rarely recovered during necropsy because they die after mating. On the other hand, females of S. muris and S. obvelata are difficult to differentiate since they resemble each other and are said to differ only in the location of the vulva. The vulva of S. muris is slightly posterior to that of S. obvelata (Farrar et al., 1994). However, this morphological difference is difficult to determine, hence not very reliable and not well described. Eggs of A. tetraptera are spindle-shaped (Ooi et al., 1994). Both S. muris and S. obvelata eggs are banana-shaped with only slight differences in the size, and it necessitates measurement of the eggs and examination of adult worms before one can identify the species. Also, in those cases where only fragments of worms are recovered, identification of the parasite by its morphology is very difficult, if not impossible. Thus, there is a need for an easier and more reliable alternative method for the identification and differentiation of the pinworms. In this study, we employed molecular techniques for the identification and differentiation of pinworms. Moreover, the ribosomal DNA sequences of pinworms have not yet been reported. Thus, the purpose of this study is to amplify the rDNA region, particularly the second internal transcribed spacer region for the differentiation of S. obvelata, S. muris and A. tetraptera.
primers NC5 (forward; 50 -GTAGGTGAACCTGCGGAAGGATCATT-30 ) and NC2 (reverse; 50 TTAGTTTCTTTTCCTCCGCT-30 ). Reactions were carried out in 50 ml reaction volume with 10 mM Tris–HCl, pH 8.4; 50 mM KCl; 3.0 mM MgCl; 250 mM each of dNTP; 20–50 pmol of each primer with 2 U Taq Polymerase (Protech, Taiwan) and worm DNA under the following conditions: 94 8C, 30 s (denaturation); 60 8C, 30 s (annealing); 72 8C, 30 s (extension) for 30 cycles followed by a final extension at 72 8C for 5 min using a thermal cycler (Minicycler, MJ Research). The PCR fragments were separated electrophoretically on 2% agarose gels, visualized using ethidium bromide and photographed. The length of PCR products were estimated using a 100 bp ladder marker (Genemark Technology Co., Ltd., Taiwan). 2.3. Sequencing and alignment of PCR products
S. obvelata and A. tetraptera from mice and S. muris from rats were obtained from the caecum and colon of the animals at necropsy. Briefly, the caecum and colon were removed and opened longitudinally in a petri dish with 0.85% saline solution. Worms were then collected and washed in physiologic saline and identified under light and dissection microscope based on the classification by Pinto et al. (2001) and by the descriptions made by Ooi et al. (1994) and Farrar et al. (1994).
Purified PCR products were sequenced in both directions using universal primers NC5 (forward; 50 -GTAGGTGAACCTGCGGAAGGATCATT-30 ) and NC2 (reverse; 50 -TTAGTTTCTTTTCCTCCGCT-30 ) amplifying the ITS-1, 5.8S and ITS-2 rDNA regions. Sequencing was carried out by a commercial sequencing company using an automated sequencer machine (ABI 3730, Applied Biosystems). To verify the sequence, sequencing was done thrice on individual PCR reactions. The forward and reverse sequences were then compared using the Align2 program (BLAST), aligned using CLUSTAL W and then corrected visually. The 50 and 30 ends of the ITS-1, 5.8S and ITS-2 sequences were determined by comparison to that of Enterobius vermicularis (DQ8322281, AB222054– AB222059) and Caenorhabditis elegans (X03680). A consensus sequence was generated for each species and were aligned using CLUSTAL W. Pairwise p-distances were calculated for the ITS-1, 5.8S and ITS-2 and for the whole rDNA region between species with the program MEGA 3.1 (Kumar et al., 2004).
2.2. Extraction and amplification of rDNA by PCR
2.4. PCR-RFLP and species-specific primer design
Extraction of genomic DNA was performed on individual worms. The worms were first homogenized using a sterile plastic homogenizer and genomic DNA was extracted using the Tissue Genomic DNA Extraction System (Viogene1) according to the manufacturer’s protocol. DNA was then stored at 20 8C until use. The rDNA region comprising the ITS-1, 5.8S gene and ITS-2 was amplified by PCR using universal
Purified NC5-NC2 PCR products (7 ml) were digested directly with 8–10 units (1 ml) of each restriction endonuclease (AluI, RsaI; Toyobo) in a volume of 10 ml at 37 8C for 4–6 h according to manufacturer’s protocol. Restriction fragments were then separated on 3% agarose gels, stained with ethidium bromide and then photographed (Fig. 1). Primers were designed to unique sequences of the 50 end of the ITS-2 region of each species and a reverse
2. Materials and methods 2.1. Parasites
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Fig. 1. (A) PCR-RFLP of NC5-NC2 PCR products using AluI. M: 100 bp ladder marker, Lanes 1 and 2: S. obvelata, Lanes 3 and 4: S. muris, and Lanes 5 and 6: A. tetraptera. (B) PCR-RFLP of NC5-NC2 PCR products using RsaI. M: 100 bp ladder marker, Lanes 1 and 2: S. obvelata, Lanes 3 and 4: S. muris, and Lanes 5 and 6: A. tetraptera.
primer was also designed for A. tetraptera using Vector NTI Suite1 software. The designed primers for both Syphacia spp. were used with reverse primer NC2. The PCR condition used was as previously described for NC5-NC2. Primers were evaluated for their specificity by using genomic DNA from S. obvelata, S. muris, A. tetraptera and Toxocara canis. Amplicons generated by the specific primers were then sequenced using the corresponding specific primers. 3. Results ITS-1, 5.8S and the ITS-2 sequences were determined and characterized for S. obvelata, S. muris and A. tetraptera. ITS-1 sequences were 314–456 bp while the length of the ITS-2 ranged from 273 to 419 bp. The 5.8S gene was 157 bp (full sequence) for the three species. The G + C content of the three regions for S. obvelata, S. muris and A. tetraptera were 41.26%, 48.12%, and 43.02%, respectively. Intra-individual sequence polymorphism, defined herein as the presence of two nucleotide bases in one alignment position, was detected in one site of the first internal transcribed region (ITS-1) of A. tetraptera. Polymorphisms were not detected in any region of the rDNA of S. obvelata and S. muris. Pairwise p-distance between species using the three rDNA regions ranged from 0.369 to 0.488. Sequence variation between species for the ITS-1 (0.454–0.604) and ITS-2 (0.456–0.593) were higher compared to the 5.8S region (0.076–0.096). Some regions of alignment were conserved while smaller regions of the ITS-1 and ITS-2 were similar for the three species. However, there are also some unique smaller
regions in the ITS-2 region that could be used as genetic markers. RFLP using AluI and RsaI allowed the delineation of the three species, S. obvelata, S. muris and A. tetraptera. As expected, digestion of NC5-NC2 PCR products with AluI produced 3 fragments (320, 244, and 179 bp) for S. obvelata, 2 fragments (561 and 252 bp) for S. muris, and 4 fragments for A. tetraptera (373, 353, 255, and 212 bp). Digestion with RsaI produced 2 fragments (450 bp and 339 bp) for S. obvelata while 2 fragments of 620 bp and 290 bp were produced for S. muris. Strong bands of 683 bp and 273 bp were produced for A. tetraptera, while fainter bands were detected at 196 and 100 bp. Primers Sof (50 -ACAAATTAAAGTTGTCGACTGACTG-30 ), Smf (50 -CCTATGACGATGGCATGTTC30 ), and Aspf (50 -ATACTCTTTAACGCATACAC-30 ) were designed to unique 50 regions of the ITS-2 of S. obvelata, S. muris, and A. tetraptera, respectively (Fig. 2). A reverse primer AspR(50 -TGCGGCCTACAGTAAAAAGC-30 ) was also designed for A. tetraptera. Sof-NC2 succesfully amplified 350 bp segment from S. obvelata but not from S. muris, A. tetraptera and T. canis (Fig. 2). An amplicon of 400 bp was amplified by primers Smf-NC2 from S. muris but not from S. obvelata, A. tetraptera and T. canis. Primers Aspf-AspR amplified 500 bp pcr product from A. tetraptera but not from S. obvelata, S. muris and T. canis. The PCR products amplified by primers from the genomic DNA of the corresponding species were sequenced with the corresponding specific primer and were proven to be ITS-2 sequences of the appropriate species.
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Fig. 2. Results of amplification by PCR of genomic DNA from S. obvelata (Lane 1), S. muris (Lane2), A. tetraptera (Lane 3) and T. canis (Lane 4), using the following primer sets: Sof-NC2 (panel A); Smf-NC2 (panel B); Aspf-AspR (panel C). M: Marker 100 bp ladder.
4. Discussion Molecular techniques have been extensively used for the identification and phylogenetic analysis of nematodes (Zhu et al., 1998b; Morales-Hojas et al., 2001; Nakano et al., 2006; Li et al., 2007). In particular, the amplification of the nuclear ribosomal region has been employed for differentiation of morphologically similar and/or closely related organisms (Jacobs et al., 1997; Zhu et al., 2007). However, in rodent pinworms, only the mitochondrial cytochrome c oxidase gene sequences have been reported (Okamoto et al., 2007) and that the nuclear ribosomal DNA sequences of rodent pinworms have not yet been investigated. To differentiate the morphologically similar pinworms of the common laboratory rodents, such as S. obvelata and S. muris, we amplified and sequenced the region spanning the internal transcribed spacer 1 (ITS-1), 5.8S, and ITS-2 of the nuclear ribosomal DNA followed by designing species-specific primers for future use in identification of the worms. We also showed that S. obvelata, S. muris and A. tetraptera differ in their ITS-1, 5.8S, and ITS-2 sequences and that the morphologically similar, S. obvelata and S. muris, can be identified and delineated based on their rDNA sequences. This is the first report of the ITS-1, 5.8S, and ITS-2 of the rDNA of the three aforementioned rodent pinworm species. rDNA sequences reported in this study have been submitted to Genbank and given accession numbers EU263105– EU263107. Sequence polymorphism and intraspecies variation were not detected in any of the rDNA regions of both Syphacia spp. In A. tetraptera, however, one site of polymorphism (e.g. presence of two nucleotide bases in one alignment position) was detected in the first internal transcribed spacer. Polymorphism has been detected in
the internal transcribed spacer regions of other species of parasitic nematodes (Jacobs et al., 1997). Genetic variation between species was higher in ITS1 and ITS-2 regions than in the 5.8S. This is expected because of the highly conserved nature of the 5.8S gene (Zhu et al., 1998a). Moreover, this implies that as in other nematodes (Zhu et al., 1998b; Li et al., 2007), the internal transcribed spacers can also be used in the identification and differentiation of closely related and/ or morphologically similar species of pinworms. Species-specific primers were hence designed to unique regions of the ITS-2 for each of the three species. As expected, primer sets Sof-NC2, Smf-NC2 and AspfAspR only amplified genomic DNA from S. obvelata, S. muris, and A. tetraptera, respectively, without amplifying gDNA from the other pinworm species tested. Digestion of PCR products generated by the universal primers NC5-NC2 with AluI or RsaI produced distinct banding patterns that can delineate the three species. The use of species-specific primers along with PCRRFLP can be used for a more rapid and more reliable identification of rodent pinworms. These molecular methods can be particularly useful for use on female worms or when only fragments of the worm are available and in other cases in which identification based on the morphology is difficult. These molecular techniques could also be used to identify the pinworm species using only the eggs of the parasite. Eggs of Syphacia spp. can be collected from the perianal region using cellophane tape technique described by Sato et al. (1995) and A. tetraptera eggs by flotation technique (Ooi et al., 1994). DNA can then be extracted from the eggs and the methods described for worm samples can be employed for the identification of the pinworm species. In conclusion, the present study describes molecular techniques for the identification and differentiation of
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the common rodent pinworms. The use of speciesspecific primers along with PCR-RFLP would be helpful for taxonomists, phylogeneticists, and for researchers whenever these pinworms and their effects are under investigation. These would also serve as useful tools for the rapid identification of pinworms in laboratory and wild rodents. Acknowledgement This work was partially supported by the ATU Project Grant for NCHU from the Ministry of Education, Taiwan. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j. plantsci.2004.08.011. References Baker, D., 1998. Natural pathogens of laboratory mice, rats, and rabbits and their effects on research. Clin. Microbiol. Rev. 11, 231–266. Bazzano, T., Restel, T.I., Pinto, R.M., Gomes, D.C., 2002. Patterns of infection with nematodes Syphacia obvelata and Aspicularis tetraptera in conventionally maintained laboratory mice. Mem. Inst. Oswaldo Cruz 97, 847–853. Beattie, G.M., Baird, S., Lannom, R., Slimmer, S., Jensen, F.C., Kaplan, N.O., 1980. Induction of lymphoma in athymic mice: a model for study of the human disease. Proc. Natl. Acad. Sci. 77, 4971–4974. Bugarski, D., Joveie, G., Katic-Radivojevic, S., Petakov, M., Krstic, A., Stojanovic, N., Milenkovic, P., 2006. Hematopoietic changes and altered reactivity to IL-17 in Syphacia obvelata-infected mice. Parasitol. Int. 55, 91–97. Perriat Perec-Matysiak, A., Okulewicz, A., Hildebrand, J., Zalesny, G., 2006. Helminth parasites of laboratory mice and rats. Wiad. Parasitol. 52, 99–102. Farrar, P.L., Wagner, J.E., Kagiyama, N., 1994. Syphacia spp. In: Waggie, K., Kagiyama, N., Allen, A., Nomura, T. (Eds.), Manual of Microbiologic Monitoring of Laboratory Animals. second ed. National Institutes of Health, United States of America, pp. 219–224. Jacobs, D.E., Zhu, X.Q., Gasser, R.B., Chilton, N.B., 1997. PCRbased methods for the identification of potentially zoonotic ascaridoid parasites of the dog, fox and cat. Acta Trop. 68, 191–200. Kumar, S., Tamura, K., Nei, M., 2004. MEGA 3 Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief. Bioinf. 5, 150–163.
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