Development of PCR/dot blot assay for specific detection and differentiation of taeniid cestode eggs in canids Maria Teresa Armua-Fernandez a , Nariaki Nonaka b , Tatsuya Sakurai a , Seita Nakamura a , Bruno Gottstein c , Peter Deplazes d , Isaac G.K. Phiri e , Ken Katakura a , Yuzaburo Oku a, ⁎ a Laboratory of Parasitology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Kita 18 Nishi 9, Sapporo, 060-0818, Japan b Laboratory of Veterinary Parasitic Diseases, Department of Veterinary Sciences, Faculty of Agriculture, University of Miyazaki, Gakuen-kibanadai-nishi-1-1, Miyazaki, 889-2192, Japan c Institute of Parasitology, University of Bern, Länggass-Strasse 122, CH-3012 Bern, Switzerland d Institute for Parasitology, Zürich University, Winterthurer-Strasse 266a, CH-8057 Zürich, Switzerland e Department of Clinical Studies, Laboratory of Helminthology, Samora Machel School of Veterinary Medicine, University of Zambia, P.O. Box 32379, Lusaka, Zambia abstract article info Article history: Received 18 September 2010 Received in revised form 17 November 2010 Accepted 17 November 2010 Available online 25 November 2010 Keywords: Taeniid cestode eggs Species-specific oligonucleotide probes PCR/dot blot assay We report the development of a colourimetric PCR/dot blot assay targeting the mitochondrial gene NADH dehydrogenase subunit 1 (nad1) for differential diagnosis of taeniid eggs. Partial sequences of the cestode nad1 gene were aligned and new primers were designed based on conserved regions. Species-specific oligonucleotide probes (S-SONP) for canine taeniid cestodes were then designed manually based on the variable region between the conserved primers. Specifically, S-SONP were designed for the Taenia crassiceps, T. hydatigena, T. multiceps, T. ovis, T. taeniaeformis, Echinococcus granulosus (genotype 1), E. multilocularis and E. vogeli. Each probe showed high specificity as no cross-hybridisation with any amplified nad1 fragment was observed. We evaluated the assay using 49 taeniid egg-positive samples collected from dogs in Zambia. DNA from 5 to 10 eggs was extracted in each sample. Using the PCR/dot blot assay, the probes successfully detected PCR products from T. hydatigena in 42 samples, T. multiceps in 3 samples, and both species (mixed infection) in the remaining 4 samples. The results indicate that the PCR/dot blot assay is a reliable alternative for differential diagnosis of taeniid eggs in faecal samples. © 2010 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Canids, such as dogs, dingoes, foxes, wolves and jackals, harbour the adult stage of important taeniid cestode species, including Echinococcus granulosus, E. multilocularis, Taenia ovis, T. multiceps and T. hydatigena. Whereas the dangers caused by larval stages of Echinococcus species to public health are well known [1], the potential risk associated with zoonotic infection by metacestodes of several Taenia spp. such as T. multiuceps (Coenurus cerebralis) [2] and T. crassiceps (Cysticecus longicollis) [3] is poorly clarified. In fact, several Taenia species have been reported to be highly prevalent in many countries including Uruguay [4], Ethiopia [5], and Italy [6] where tons of carcasses and offal are discarded every year due to infection of domestic livestock by taeniid larvae. The lack of accurate diagnostic methods for Taenia species differentiation in live canids further hinders our understanding of the biology and host–parasite interaction of these parasites. Since canids can harbour several species of Taenia and Echinococcus simultaneously, developing a method for detecting and distinguishing between taeniid eggs present in faeces is considered to be essential. While the detection of coproantigens by ELISA (coproELISA) [7,8] and PCR (copro-DNA) [9] had previously been used to accurately diagnose infection by Echinococcus spp., analogous methods for distinguishing between species of Taenia in canids have not yet been developed. Although Gasser and Chilton [10] and Trachsel et al. [11] successfully discriminated Taenia spp. by PCR-RFLP, their methods had not been used in survey studies. The PCR/dot blot assay is a widely used hybridisation technique that has been applied to the identification and genotyping of pathogens such as Mycobacterium tuberculosis [12], Chlamydia psittaci [13], and Echovirus [14]. The simplicity of this hybridisation assay enables simultaneous and rapid screening of several samples and is capable of species differentiation using species-specific oligonucleotide probes. Lavikainen et al. [15] reported that Taenia spp. can be differentiated based on the polymorphisms of the cytochrome c oxidase subunit 1(cox1) gene and NADH dehydrogenase subunit 1 (nad1) gene sequences. Among these two genes, we chose nad1 as a candidate for oligonucleotide probes design on the basis of the higher variability range [16]. In addition much more sequences of this gene are currently registered in the GenBank database compared to other genes studied to date. Parasitology International 60 (2011) 84–89 ⁎ Corresponding author. Tel.: +81 11 706 5198; fax: +81 11 706 5196. E-mail address: oku@vetmed.hokudai.ac.jp (Y. Oku). 1383-5769/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2010.11.005 Contents lists available at ScienceDirect Parasitology International journal homepage: www.elsevier.com/locate/parint