New genotyping method for the causative agent of crayfish plague (Aphanomyces astaci) based on whole genome data.

J Invertebr Pathol

Centre for Environment, Fisheries and Aquaculture Science (Cefas), Barrack Road, DT4 8UB Weymouth, UK. Electronic address:

Published: July 2018

AI Article Synopsis

  • The oomycete Aphanomyces astaci is responsible for crayfish plague, a major disease affecting European freshwater crayfish, likely introduced from North American crayfish 150 years ago.
  • Researchers have classified five genotypes of A. astaci using RAPD-PCR, which is crucial for understanding the disease's epidemiology in Europe.
  • This study presents a new genotyping method that can analyze DNA from clinical crayfish samples, successfully distinguishing four out of five genotypes, enhancing the tools available for studying and monitoring crayfish plague.

Article Abstract

The oomycete Aphanomyces astaci causes crayfish plague, the most important disease of European freshwater crayfish species. Presumably introduced into Europe 150 years ago with the import of North American crayfish, A. astaci is highly pathogenic to European crayfish species. Five genotypes (A, B, C, D, and E) have been defined based on random amplified polymorphic DNA analysis (RAPD-PCR) from A. astaci pure cultures. The distinction of genotypes is an essential tool to conduct molecular epidemiological studies on crayfish plague and it has been used to clarify and better understand the history and spread of this disease in Europe. Whereas RAPD-PCR requires DNA from pure culture isolates, the development of genotyping tools that can be applied to DNA extracted from clinical samples allows a much wider application of genotyping studies, including revisiting historic samples. In this study, we present a new approach that adds to currently available methods for genotyping A. astaci strains directly from clinical crayfish samples. Whole-genome sequencing of A. astaci strains representing all currently known genotypes was employed, genomic regions unique to the respective genotype identified, and a PCR-based genotyping assay designed, which focuses on the presence/absence of PCR product after amplification with the genotype-specific primers. Our diagnostic methodology was tested using DNA extracts from pure A. astaci cultures, other Aphanomyces species and additional oomycetes, samples from a recent Italian crayfish plague outbreak and additional historical samples available in the Centre for Environment, Fisheries and Aquaculture Science laboratory. The new markers were reliable for pure culture and clinical samples from a recent outbreak and successfully discriminated genotype A, B, D, and E. The marker for genotype C required an additional sequencing step of the generated PCR product to confirm genotype.

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Source
http://dx.doi.org/10.1016/j.jip.2018.06.002DOI Listing

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