Re-examination of the prevalence of Aphanomyces astaci in North American crayfish populations in Central Europe by TaqMan MGB real-time PCR

Abstract

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.

Continental-wide distribution of crayfish species in Europe: update and maps

“…So far, however, the species apparently did not spread further, either upstream to the Czech Republic or inland into Slovakia. Although original analyses did not suggest the infection by A. astaci (Petrusek and Petrusková, 2007), subsequent use of a more sensitive molecular method detected presence of the pathogen in this population (Kozubíková et al, 2011).…”

Invasive crayfish and crayfish plague on the move: first detection of the plague agent Aphanomyces astaci in the Romanian Danube

“…The identity of Aphanomyces astaci was further confirmed by amplifying and sequencing a 529 bp long ITS fragment (including ITS1 as well as ITS2) from 6 DNA isolates representing both host crayfish species, according to protocol used by , with primers 42 and 640 designed to be specific for A. astaci (but see Kozubíková et al 2011b for discussion on their limitation). The PCR products were sequenced in the forward direction using the primer 42, and resulting sequences (GenBank accession numbers JN713915−JN713917) were aligned in Kozubíková et al 2011a).…”

“…Possible real-time PCR inhibition was checked for by comparison of results obtained from concentrated and diluted samples. In the absence of inhibition, with an acceptance of 15% variation, the difference in cycle threshold (C t ) values should be 2.32 ± 0.35 and 3.32 ± 0.48 for the 5 and 10× dilutions, respectively, when compared to undiluted DNA, (see Kozubíková et al 2011b for more details). However, as many samples, particularly from Orconectes limosus, yielded results below LOQ, a quantitative comparison could not be made.…”

“…Recent results show that this method offers higher sensitivity than any other presently available, and it is the preferred method for screening populations of North American crayfish for carrier status (Tuffs & Oidtmann 2011). Furthermore, this method leads to lower error rates in the detection of Apha nomyces astaci than alternatives based on conventional PCR (Kozubíková et al 2011b). …”

“…During the last several years, molecular diagnostic tools employing conventional PCR and real-time PCR methods for the specific detection of Aphanomyces astaci have been developed, and these have accelerated and improved the detection and identification of the crayfish plague agent , Hochwimmer et al 2009, Vrålstad et al 2009). The carrier status of symptom-free North American crayfish is also evaluated using these tools , Kozubíková et al 2009, 2011b, Vrålstad et al 2009, 2011, Skov et al 2011.…”