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Multiplex PCR system for identifying the carnivore origins of faeces for an epidemiological study on Echinococcus multilocularis in Hokkaido, Japan

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Abstract

A multiplex PCR system was developed to identify the carnivore origins of faeces collected in Hokkaido, Japan, for epidemiological studies on Echinococcus multilocularis. Primers were designed against the D-loop region of mitochondrial DNA. Two separate primer mixtures (mix 1, specific forward primers to fox, raccoon dog and dog, and a universal reverse primer [prH]; and mix 2, specific forward primers to cat, raccoon and weasels and prH) were used so that the PCR products (160 bp, fox and cat; 240 bp, raccoon dog and raccoon; and 330 bp, dog and weasel) were distinguished by size. The multiplex PCR exhibited no cross-reactivity between carnivore species and did not amplify DNA from rodent prey. When 270 field-collected faeces were examined, 250 showed single PCR products belonging to specific target sizes, suggesting successful carnivore identification for 92.6% of samples. Taeniid eggs were detected in 11.1% of samples and coproantigen in 30.4%; whereas the prevalences of taeniid eggs and coproantigen were 12.9% and 34.0% in fox faeces, and 0% and 26.3% in cat faeces, respectively. These results suggest that the prevalence in different target animals can be evaluated individually and precisely using multiplex PCR system.

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Acknowledgements

The authors acknowledge Dr. Hitoshi Suzuki in Graduate School of Environmental Earth Science, Hokkaido University, for providing DNA materials from rodents in the field. We would like to thank Drs. Bruno Gottstein (University of Bern), Peter Deplazes (University of Zurich), Alexander Mathis (University of Zurich), Francis Raoul (University of Franche-Comte) and Jean M. Bart (Hospital Minjoz, France) for supplying us parasite samples. We are also grateful to Dr. Guo Zhihong in University of Miyazaki, and the members of the Laboratory of Parasitology, Graduate School of Veterinary Medicine, Hokkaido University, for their valuable support. This work was supported by the Japan Society for the promotion of Science (grant no. 15380205) and by the Ministry of Health, Labour and Welfare, Japan (grant for "The control of emerging and reemerging diseases in Japan").

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Authors and Affiliations

  1. Laboratory of Veterinary Parasitic Diseases, Department of Veterinary Sciences, Faculty of Agriculture, University of Miyazaki, Gakuen-Kihanadai Nishi 1-1, Miyazaki, 889-2192, Japan

    Nariaki Nonaka

  2. Laboratory of Parasitology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Kita-ku Kita 18 Nishi 9, Sapporo, Hokkaido, 060-0818, Japan

    Takafumi Sano, Takashi Inoue, Maria Teresa Armua, Ken Katakura & Yuzaburo Oku

  3. Asahikawa Municipal Asahiyama Zoological Park & Wildlife Conservation Center, Kuranuma, Higashi Asahikawa-cho, Asahikawa, Hokkaido, 078-8205, Japan

    Daisuke Fukui

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  1. Nariaki Nonaka
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  2. Takafumi Sano
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  3. Takashi Inoue
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  4. Maria Teresa Armua
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  5. Daisuke Fukui
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  6. Ken Katakura
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  7. Yuzaburo Oku
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Correspondence to Nariaki Nonaka.

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Nonaka, N., Sano, T., Inoue, T. et al. Multiplex PCR system for identifying the carnivore origins of faeces for an epidemiological study on Echinococcus multilocularis in Hokkaido, Japan. Parasitol Res 106, 75–83 (2009). https://doi.org/10.1007/s00436-009-1629-0

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