Development of Duplex PCR Assay for Detection and Differentiation of Typical and Atypical Melissococcus plutonius strains

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  • ARAI Rie
    Saitama Prefectural Chuo Livestock Hygiene Service Center, 107–1 Besshocho, Kita-ku, Saitama, Saitama 331–0821, Japan The United Graduate School of Veterinary Sciences, Gifu University, 1–1 Yanagido, Gifu, Gifu 501–1193, Japan
  • MIYOSHI-AKIYAMA Tohru
    Department of Infectious Diseases, National Center for Global Health and Medicine, 1–21–1 Toyama, Shinjuku-ku, Tokyo 162–8655, Japan
  • OKUMURA Kayo
    Department of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, 11 Nishi 2-sen, Inada-cho, Obihiro, Hokkaido 080–8555, Japan
  • MORINAGA Yuiko
    Fukuoka-Chuo Prefectural Institute of Animal Health, 4–14–5 Hakozakifuto, Higashi-ku, Fukuoka, Fukuoka 812–0051, Japan
  • WU Meihua
    Graduate School of Life and Environmental Sciences, University of Tsukuba, 1–1–1 Tennodai, Tsukuba, Ibaraki 305–8572, Japan
  • SUGIMURA Yuya
    Honey Bee Research Unit, Animal Breeding and Reproduction Research Division, NARO Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization, 2 Ikenodai, Tsukuba, Ibaraki 305–0901, Japan
  • YOSHIYAMA Mikio
    Honey Bee Research Unit, Animal Breeding and Reproduction Research Division, NARO Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization, 2 Ikenodai, Tsukuba, Ibaraki 305–0901, Japan
  • OKURA Masatoshi
    Bacterial and Parasitic Diseases Research Division, National Institute of Animal Health, National Agriculture and Food Research Organization, 3–1–5 Kannondai, Tsukuba, Ibaraki 305–0856, Japan
  • KIRIKAE Teruo
    Department of Infectious Diseases, National Center for Global Health and Medicine, 1–21–1 Toyama, Shinjuku-ku, Tokyo 162–8655, Japan
  • TAKAMATSU Daisuke
    The United Graduate School of Veterinary Sciences, Gifu University, 1–1 Yanagido, Gifu, Gifu 501–1193, Japan Bacterial and Parasitic Diseases Research Division, National Institute of Animal Health, National Agriculture and Food Research Organization, 3–1–5 Kannondai, Tsukuba, Ibaraki 305–0856, Japan

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  • Development of Duplex PCR Assay for Detection and Differentiation of Typical and Atypical <i>Melissococcus plutonius</i> strains

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説明

Melissococcus plutonius is the causative agent of an important honeybee disease, European foulbrood (EFB). In addition to M. plutonius strains with typical characteristics (typical M. plutonius), we recently reported the presence of atypical M. plutonius, which are phenotypically and genetically distinguished from typical M. plutonius. Because typical and atypical M. plutonius may have different pathogenic mechanisms, differentiation of these two types is very important for diagnosis and more effective control of EFB. In this study, therefore, a duplex PCR assay was developed to detect and differentiate typical and atypical M. plutonius rapidly and easily. On the basis of the results of comparative genomic analyses, we selected Na+/H+ antiporter gene and Fur family transcriptional regulator gene as targets for detection of typical and atypical strains, respectively, by PCR. Under optimized conditions, the duplex PCR system using the designed primers successfully detected and differentiated all typical and atypical M. plutonius strain/isolates tested, while no product was generated from any other bacterial strains/isolates used in this study, including those isolated from healthy honeybee larval guts. Detection limits of the PCR were 50 copies of chromosome/reaction for both types, and it could detect typical and atypical M. plutonius directly from diseased honeybee larvae. Moreover, the duplex PCR diagnosed mixed infections with both M. plutonius types more precisely than standard culture methods. These results indicate that the duplex PCR assay developed in this study is extremely useful for precise diagnosis and epidemiological study of EFB.

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