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

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概要

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• 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.

• 公益社団法人 日本獣医学会の論文

著者

• Kirikae Teruo

  Department Of Infections Diseases And Tropical Medicine Research Institute International Medical Cen

• Okumura Kayo

  Department Of Infectious Diseases Research Institute International Medical Center Of Japan

• Yoshiyama Mikio

  Honeybee Research Group, Animal Breeding and Reproduction Research Team, National Institute of Lives

• Miyoshi-Akiyama Tohru

  Department of Microbiology and Immunologu, Tokyo Women's Medical College

• KIRIKAE Teruo

  Department of Infectious Diseases, National Center for Global Health and Medicine

• ARAI Rie

  Saitama Prefectural Chuo Livestock Hygiene Service Center

• MORINAGA Yuiko

  Fukuoka-Chuo Prefectural Institute of Animal Health

• WU Meihua

  Graduate School of Life and Environmental Sciences, University of Tsukuba

• 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

• OKURA Masatoshi

  Bacterial and Parasitic Disease Research Division, National Institute of Animal Health, National Agriculture and Food Research Organization

• TAKAMATSU Daisuke

  The United Graduate School of Veterinary Sciences, Gifu University

• OKUMURA Kayo

  Department of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine

• MIYOSHI-AKIYAMA Tohru

  Department of Infectious Diseases, National Center for Global Health and Medicine

• 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

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