In Central Europe invasive North American crayfishes are carriers of the oomycete Aphanomyces astaci, which causes crayfish plague. This lethal disease currently represents one of the major threats to native European crayfishes. We used molecular methods-species--specific amplification and sequencing of the pathogen DNA--to investigate the prevalence of individuals latently infected with A. astaci in 28 populations of two invasive American crayfish species (6 of the signal crayfish [Pacifastacus leniusculus] and 22 of the spiny-cheek crayfish [Orconectes limosus]) in the Czech Republic. The pathogen occurred in 17 investigated populations. We recorded a high variation in positive reactions, ranging from 0% to 100%, in populations of O. limosus. In P. leniusculus, however, only one individual out of 124 tested positive for the pathogen. There was a clear relationship between the water body type and pathogen prevalence in O. limosus. Infection ratios in isolated standing waters were usually low, whereas in running waters, pathogen prevalence often exceeded 50%. Other evaluated characteristics of potential plague pathogen carriers (size, sex, and the presence of melanized spots in the cuticle) seemed to be unrelated to infection. Our data suggest that in contrast to other European countries, O. limosus seems to be the primary reservoir of crayfish plague in the Czech Republic. Although all populations of alien American crayfishes may be potential sources of infections and should be managed as such, knowledge on the prevalence of the plague pathogen at various localities may allow managers to focus conservation efforts on the most directly endangered populations of native crayfishes.

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We applied quantitative TaqMan minor groove binder real-time polymerase chain reaction (PCR) on DNA isolates from soft abdominal cuticle of 460 North American crayfish Orconectes limosus and Pacifastacus leniusculus, previously tested for Aphanomyces astaci presence by conventional semi-nested PCR. Both approaches target the internal transcribed spacers of the pathogen nuclear ribosomal DNA, but apply different specific sequence motifs and technologies. The real-time PCR approach seems to provide higher sensitivity; the number of crayfish that tested positive increased from 23 to 32%, and 10 additional crayfish populations were indicated as hosting the disease agent. However, the vast majority of newly recorded positives contained very low agent levels, from 5 to 50 PCR-forming units. An isolate producing a false positive result by the semi-nested PCR (apparently undescribed Aphanomyces related to A. astaci) remained negative using the real-time PCR. The present study shows that previous results based on the seminested PCR were not substantially influenced by false positives but might have suffered from some false negatives at low agent levels. Combining alternative methods may therefore provide more reliable conclusions on the pathogen's presence. Further, we found positive correlation between the prevalence of infection carriers in American crayfish populations and the average amounts of A. astaci DNA detected in infected local crayfish individuals.

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