Hunting the crayfish plague disease agent in water sources – challenges and possibilities

The specialized North American crayfish parasite and oomycete Aphanomyces astaci causes crayfish plague in European crayfish. Infectious A. astaci zoospores are released from American carrier crayfish and from diseased or dead European crayfish. The A. astaci spore quantity in the ambient water of crayfish will therefore largely reflect disease outbreaks in European crayfish or carrier status in American crayfish. During the past few years, molecular methods detecting A. astaci directly from crayfish tissues has become widely accepted. Now, the first studies employing A. astaci specific qPCR directly from water samples are approaching. Monitoring waterways could facilitate early warning of A. astaci infections, improve risk assessments, and reveal of infection reservoirs. It might also serve as a supplementary tool for declaring crayfish stocks and water bodies infection free in nature and aquaculture. Over the past three years, we have worked on this topic in a Nordic collaborative project. Here, we present methodological experiences e.g. sampling volumes, filtration methods, and subsequent molecular analyses. Possibilities and challenges connected to the transition from laboratory, via aquaria models, to natural environments are discussed. Furthermore, unknown diversity of oomycetes resembling A. astaci in the target DNA motif challenges the reliability of qPCR results, both from crayfish and natural environmental samples. We have therefore identified means that increase the probability of distinguishing between possibly true and false positives. These include qPCR optimizations, and use of a decision support tool that evaluates the qPCR result reliability on the basis of linearity (R2) and amplification efficiency of the PCRs.