Detection of parasitizing coccidia and determination of host crane species, sex and genotype by faecal DNA analysis

Authors

  • H. HONMA,

    1. Laboratory of Sustainable Environmental Biology, Graduate School of Agricultural Science, Tohoku University, 232-3 Naruko-onsen Yomogida, Osaki, Miyagi 989-6711, Japan
    2. Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan
    3. Laboratory of Malariology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan
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  • Y. SUYAMA,

    1. Laboratory of Forest Ecology, Graduate School of Agricultural Science, Tohoku University, 232-3 Naruko-onsen Yomogida, Osaki, Miyagi 989-6711, Japan
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  • Y. NAKAI

    1. Laboratory of Sustainable Environmental Biology, Graduate School of Agricultural Science, Tohoku University, 232-3 Naruko-onsen Yomogida, Osaki, Miyagi 989-6711, Japan
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Y. Nakai, Fax: +81 0229 84 7391; E-mail: nakai@bios.tohoku.ac.jp

Abstract

In Japan, the three main crane species are the endangered red-crowned crane (Grus japonensis) inhabiting Hokkaido, the northernmost island of Japan; the vulnerable hooded crane (Grus monacha); and the vulnerable white-naped crane (Grus vipio). Both the hooded and white-naped cranes migrate in winter to Izumi in Kyushu, the southern island of Japan. In this study, we investigated the cranes and their coccidian parasites, through a targeted molecular approach using faecal DNA to develop a noninvasive method for infectious disease research. To determine the origin of noninvasively collected faecal samples, host species were identified by sequencing a region of approximately 470 bp of the mitochondrial 16S ribosomal RNA gene in the faecal DNA. Furthermore, to avoid sample redundancy, individual determination was performed by fragment analysis using microsatellite and sex-linked markers. For microsatellite genotyping, previously reported markers and markers isolated in this study were examined, and seven loci for red-crowned cranes, eight for hooded cranes and six for white-naped cranes displayed polymorphisms. A low error rate was demonstrated by comparing microsatellite data generated from faecal DNA samples with that generated from feather DNA samples, indicating a high reliability. Polymerase chain reaction–based capillary electrophoresis (PCR-CE), employing genetic markers in the second internal transcribed spacer (ITS2) of nuclear ribosomal DNA, was employed to detect crane coccidia. The sensitivity of detection of PCR-CE using faecal DNA was inferior to that with traditional microscopy; however, our results suggest that PCR-CE can depict crane coccidia diversity with higher resolution and it is a useful tool to characterize community composition of coccidia in detail.

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