Abstract
A hybridization probe-based real-time multiplex-nested PCR system was developed for the simultaneous detection of Echinococcus multilocularis and host species directly from faecal samples. Species identification was determined by melting curve analysis. Specificity was assessed by using DNA extracted from various cestodes (E. multilocularis, Echinococcus granulosus (G1), Echinococcus ortleppi, Echinococcus canadensis (G6, G7), Taenia crassiceps, Taenia hydatigena, Taenia mustelae, Taenia pisiformis, Taenia serialis, Taenia taeniaeformis, Mesocestoides leptothylacus), carnivores (Vulpes vulpes, Vulpes corsac, Vulpes ferrilata, Canis familiaris, Felis catus, Martes foina), Microtus arvalis and Arvicola terrestris. The analytical sensitivity was 10 fg, evaluated with serially diluted DNA of E. multilocularis to 10 μl total DNA solution from E. multilocularis-negative canid faeces. Based on a comparison of 47 dog samples from China, the proportion of the E. multilocularis-positive-tested samples by the real-time multiplex-nested PCR was moderately higher (38% vs. 30%) as when tested with a previously evaluated nested PCR with a sensitivity of 70–100%, depending on the number and gravidity status of worms present in the intestine (Dinkel et al., J Clin Microbiol 36:1871–1876, 1998). To assess the epidemiological applicability of this method, 227 canid faecal samples collected in the field were analysed. This newly developed real-time multiplex-nested PCR system is a specific, sensitive and reliable method for the detection of E. multilocularis and host species in faecal samples for epidemiological purposes.
References
Bretagne S, Guillou JP, Morand M, Houin R (1993) Detection of Echinococcus multilocularis DNA in fox faeces using DNA amplification. Parasitology 106:193–199
Deplazes P, Alther P, Tanner I, Thompson RC, Eckert J (1999) Echinococcus multilocularis detection by enzyme-linked immunosorbent assay in fox, dog and cat populations. J Parasitol 85:115–121
Deplazes P, Dinkel A, Mathis A (2003) Molecular tools for studies on the transmission biology of Echinococcus multilocularis. Parasitology 127:53–61
Deplazes P, Hegglin D, Gloor S, Romig T (2004) Wilderness in the city: the urbanization of Echinococcus multilocularis. Trends Parasitol 20:77–84
Dinkel A, von Nickisch-Rosenegk M, Bilger B, Merli M, Lucius R, Romig T (1998) Detection of Echinococcus multilocularis in the definitive host: coprodiagnosis by PCR as an alternative to necropsy. J Clin Microbiol 36:1871–1876
Dinkel A, Njoroge EM, Zimmermann A, Wälz M, Zeyhle E, Elmahdi IE, Mackenstedt U, Romig T (2004) A PCR system for detection of species and genotypes of the Echinococcus granulosus-complex, with reference to the epidemiological situation in eastern Africa. Int J Parasitol 34:645–653
Dinkel A, Romig T and Mackenstedt U (2006) PCR system for the combined detection of Echinococcus multilocularis and host species from faecal samples. Deutsche Gesellschaft für Parasitologie, 22.Jahrestagung, Wien, 22.-25.02.2006
Farrell LE, Roman J, Sunquist ME (2000) Dietary separation of sympatric carnivores identified by molecular analysis of scats. Mol Ecol 9:1583–1590
Foran DR, Crooks KR, Minta SC (1997a) Species identification from scat: an unambiguous genetic method. Wildl Soc Bull 25:835–839
Foran DR, Minta SC, Heinemeyer KS (1997b) DNA-based analysis of hair to identify species and individuals for population research and monitoring. Wildl Soc Bull 25:840–847
Giraudoux P, Craig PS, Delattre P, Bao G, Bartholomot B, Harraga S, Quere JP, Raoul F, Wang Y, Shi D, Vuitton DA (2003) Interactions between landscape changes and host communities can regulate Echinococcus multilocularis transmission. Parasitology 127:121–131
Hansen MM, Jacobsen L (1999) Identification of mustelid species: otter, American mink and polecat by analysis of DNA from faecal samples. J Zoology London 247:177–181
Huettner M, Siefert L, Mackenstedt U, Romig T (2009) A survey of Echinococcus species in wild carnivores and livestock in East Africa. Int J Parasitol 39:1269–1276
Lucchini Q, Fabri E, Marucco E, Ricci S, Boitani L, Randi E (2002) Noninvasive molecular tracking of colonizing wolf (Canis lupus) packs in the western Italian Alps. Mol Ecol 11:857–868
Major M, Johnson MK, Davis WS, Kellogg TF (1980) Identifying scats by recovery of bile acids. J Wildl Manage 44:290–293
Mills LS, Pilgrim KL, Schwartz MK, McKelvey K (2000) Identifying lynx and other North American felids based on mt DNA analysis. Conserv Genet 1:285–288
Monnier P, Cliquet F, Aubert M, Bretagne S (1996) Improvement of a polymerase chain reaction assay for the detection of E. multilocularis DNA in faecal samples of foxes. Vet Parasitol 67:185–195
Nonaka N, Sano T, Inoue T, Armua MT, Fukui D, Katakura K, Oku Y (2009) Multiplex PCR system for identifying the carnivore origins of faeces for an epidemiological study on Echinococcus multilocularis in Hokkaido. Japan Parasitol Res 106(1):75–83
Palomares F, Godoy JA, Piriz A, O’Brien J, Johnson WE (2002) Faecal genetic analysis to determine the presence and distribution of elusive carnivores: design and feasibility for the Iberian lynx. Mol Ecol 11:2171–2182
Paxinos E, McIntosh C, Ralls K, Fleischer R (1997) A noninvasive method for distinguishing among canid species: amplification and enzyme restriction of DNA from dung. Mol Ecol 6:483–486
Pilgrim KL, Boyd DK, Forbes SH (1998) Testing for wolf–coyote hybridization in the Rocky Mountains using mitochondrial DNA. J Wildl Manage 62:683–689
Rishi AK, McManus DP (1987) Genomic cloning of human Echinococcus granulosus DNA: isolation of recombinant plasmids and their use as genetic markers in strain characterization. Parasitology 94:369–383
Sakai H, Nonaka N, Yagi K, Oku Y, Kamiya M (1998) Coproantigen detection in a survey of E. multilocularis infection among red foxes, Vulpes vulpes schrencki, in Hokkaido, Japan. J Vet Med Sci 60:639–641
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This work was supported by the NIH Fogarty International Center (grants number RFATW-00-002 and RO1 TW001565). The content is solely the responsibility of the authors and does not necessarily represent the official views of the Fogarty International Center or the National Institutes of Health.
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Dinkel, A., Kern, S., Brinker, A. et al. A real-time multiplex-nested PCR system for coprological diagnosis of Echinococcus multilocularis and host species. Parasitol Res 109, 493–498 (2011). https://doi.org/10.1007/s00436-011-2272-0
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DOI: https://doi.org/10.1007/s00436-011-2272-0