Inter-Research > DAO > v37 > n2 > p127-134  
Diseases of Aquatic Organisms

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DAO 37:127-134 (1999)  -  doi:10.3354/dao037127

Single and nested polymerase chain reaction assays for the detection of Microsporidium seriolae (Microspora), the causative agent of 'Beko' disease in yellowtail Seriola quinqueradiata

Andrew S. Bell1,*, Hiroshi Yokoyama2, Takashi Aoki1, Makoto Takahashi3, Keigo Maruyama3

1Department of Aquatic Bioscience, Tokyo University of Fisheries, Konan 4-5-7, Minato-ku, Tokyo 108, Japan
2Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan
3Goto Station of Japan Sea-Farming Association, Arakawa, Tamanoura-cho, Minamimatsuura-gun, Nagasaki 853-05, Japan
*Present address: The Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, Scotland, UK. E-mail:

ABSTRACT: Single and nested polymerase chain reaction (PCR) assays were developed for the detection of the microsporidian parasite Microsporidium seriolae, which is responsible for emaciation and even death in farmed Japanese yellowtail. Extremely high rDNA identities exist between this parasite and other members of the as yet unclassified genus, necessitating the design of generic, rather than species-specific primer sets. The nested PCR was several orders of magnitude more sensitive than the standard single PCRs, with visible target product amplified from as little as 0.01 pg of parasite DNA (equivalent to that extracted from a single spore). The specificity of the assays was tested against a range of potential host fishes and 6 other microsporidians infecting either fish or the musculature of their hosts. Single PCRs were found to be specific to the target genus, but the nested PCR replicated rDNA from several different microsporidian genera, limiting its utility. This study highlights problems associated with the use of the rRNA gene for PCR assays of certain microsporidians, but nevertheless provides a rapid and sensitive means for the detection of pre-spore forms not possible by current staining methods. Consequently, these assays may be employed for further studies on the portals of entry, migration to the musculature and transmission of this economically important pathogen.

KEY WORDS: Microsporidium seriolae · Microsporidia · Ribosomal DNA · Polymerase chain reaction · Fish diseases

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