Non-destructive method for detecting Aphanomyces astaci, the causative agent of crayfish plague, on the individual level.

The pathogenic oomycete Aphanomyces astaci, transmitted mainly by invasive North American crayfish, causes the crayfish plague, a disease mostly lethal for native European crayfish. Due to its decimating effects on native crayfish populations in the last century, A. astaci has been listed among the 100 worst invasive species. Importantly, detecting the pathogen in endangered native crayfish populations before a disease outbreak would provide a starting point in the development of effective control measures. However, current A. astaci-detection protocols either rely on degradation-prone eDNA isolated from large volumes of water or, if focused on individual animals, include killing the crayfish. We developed a non-destructive method that detects A. astaci DNA in the microbial biofilm associated with the cuticle of individual crayfish, without the need for destructive sampling. Efficiency of the new method was confirmed by PCR and qPCR and the obtained results were congruent with the traditional destructive sampling method. Additionally, we demonstrated the applicability of the method for A. astaci monitoring in natural populations. We propose that the new method should be used in future monitoring of A. astaci presence in endangered European native crayfish individuals as an alternative to eDNA-based monitoring.

Improved method for genotyping the causative agent of crayfish plague (Aphanomyces astaci) based on mitochondrial DNA.

Aphanomyces astaci causes crayfish plague, which is a devastating disease of European freshwater crayfish. The likely first introduction of A. astaci into Europe was in the mid-19th century in Italy, presumably with the introduction of North American crayfish. These crayfish can carry A. astaci in their cuticle as a benign infection. Aphanomyces astaci rapidly spread across Europe causing the decline of the highly susceptible indigenous crayfish species. Random amplified polymorphic DNA-PCR analysis of A. astaci pure cultures characterized five genotype groups (A, B, C, D and E). Current A. astaci genotyping techniques (microsatellites and genotype-specific regions, both targeting nuclear DNA) can be applied directly to DNA extracted from infected cuticles but require high infection levels. Therefore, they are not suitable for genotyping benign infections in North American crayfish (carriers). In the present study, we combine bioinformatics and molecular biology techniques to develop A. astaci genotyping molecular markers that target the mitochondrial DNA, increasing the sensitivity of the genotyping tools. The assays were validated on DNA extracts of A. astaci pure cultures, crayfish tissue extractions from crayfish plague outbreaks and tissue extractions from North American carriers. We demonstrate the presence of A. astaci genotype groups A and B in UK waters.

Emergence of infection with Aphanomyces invadans in fish in some main aquatic ecosystems in Zimbabwe: A threat to national fisheries production.

The first outbreak on the African continent of infection with Aphanomyces invadans (the causative agent of epizootic ulcerative syndrome) in fish was confirmed in the Chobe-Zambezi rivers in 2007. The emergence of massive outbreaks of infection with A. invadans in multiple fish species exposed serious aquatic biosecurity challenges in the Southern African region. This study investigated the incursion of infection with A. invadans in fish from the main aquatic ecosystems of Zimbabwe from 2012 to 2015 using data obtained from the Department of Livestock and Veterinary Services, Zimbabwe. In some outbreaks, fish samples were collected and tested at the University of Zambia, for confirmation by histopathology and species-specific PCR. The infection was first confirmed at Darwendale water impoundment (Mashonaland West Province) in 2012, followed by Matabeleland South Province at Mtshabezi water impoundment and Nkankezi River (both 2013). An apparent southward spread continued in 2014, with virgin outbreaks at Ntalale water impoundment (Matabeleland South Province) and Mwenezi River in Midlands Province. In 2015, inland incursion was confirmed at Dutchman's Pool in Midlands Province and further north-west at the Sanyati River Basin in Lake Kariba (Mashonaland West Province). In all outbreaks, infection with A. invadans was confirmed in seven fish species, namely the African sharptooth catfish (Clarias gariepinus, Burchell, 1822), blunt-toothed African catfish (Clarias ngamensis Castelnau, 1861), yellow belly bream (Serranochromis robustus Gunther, 1864), straight fin barb (Enteromius paludinosus Peters, 1852), dashtail barb (Enteromius poechii Steindachner, 1911), large-mouth bass (Micropterus salmoides Lac'epe'de, 1802) and the three-spot tilapia (Oreochromis andersonii Castelnau, 1861). Cases were most common in the African sharptooth catfish, with mortalities more pronounced in young fish of all species. The results suggested a gradual emergence of an intractable infection with A. invadans in fish in the main aquatic ecosystems of Zimbabwe, which may have negative impact on biodiversity conservation and aquaculture.

Aphanomyces astaci isolate from latently infected stone crayfish (Austropotamobius torrentium) population is virulent.

Aphanomyces astaci infection is the cause of crayfish plague in European crayfish. Here the virulence of an A. astaci As strain isolated from apparently healthy stone crayfish (Austropotamobius torrentium) from Slovenia was compared to that of the Psl-Puujärvi A. astaci isolate in 3 crayfish species: noble crayfish (Astacus astacus), signal crayfish (Pacifastacus leniusculus) from Finland and stone crayfish from Slovenia. All 3 crayfish species were challenged with PsI-Puujärvi A. astaci and succumbed to crayfish plague, with both noble crayfish and stone crayfish showing 100% mortality, while 25% of the signal crayfish died during the challenge. In comparison, the As-Slovenia A. astaci isolate was pathogenic for noble crayfish but not for signal crayfish or stone crayfish. This finding suggests that A. astaci virulence could be species specific and a strain from latent A. astaci infection in one native European crayfish species could be detrimental to other native European crayfish species.

Degenerate ITS7 primer enhances oomycete community coverage and PCR sensitivity to Aphanomyces species, economically important plant pathogens.

Metagenomic analysis of oomycetes through deep amplicon sequencing has been conducted primarily using the ITS6-ITS7 primer set that targets the ITS1 region. While this primer set shows a perfect match to most oomycete taxa, ITS7 contains 3 mismatches to the corresponding binding site of plant pathogens within the genus Aphanomyces. Polymerase chain reaction (PCR) efficiency differs for taxa with uneven primer matching characteristics, which may explain why previous studies have detected this genus at low abundance. To overcome the impact of these mismatches on PCR sensitivity, the mismatched nucleotides were replaced with degenerate nucleotides. Oomycete communities from 35 soil samples collected from asymptomatic and root rot diseased sites in pea fields across Alberta were analyzed simultaneously using ITS6-ITS7 and ITS6-ITS7-a.e. (modified version of ITS7) primer sets on 1 Illumina MiSeq run. The number of high-quality reads obtained by ITS6-ITS7-a.e. was more than twice that of ITS6-ITS7. The relative abundance of Pythium spp. was reduced and Aphanomyces spp. increased. Aphanomyces cf. cladogamus and Aphanomyces euteiches were the second and third most abundant species, respectively, in the pea rhizosphere using the ITS7-a.e. primer, but were rare using the ITS7 primer. These results indicate that use of ITS7-a.e. provides a more accurate picture of oomycete communities than ITS7 by enhancing PCR sensitivity to Aphanomyces.

The prevalence of Aphanomyces astaci in invasive signal crayfish from the UK and implications for native crayfish conservation.

The crayfish plague agent, Aphanomyces astaci, has spread throughout Europe, causing a significant decline in native European crayfish. The introduction and dissemination of this pathogen is attributed to the spread of invasive North American crayfish, which can act as carriers for A. astaci. As native European crayfish often succumb to infection with A. astaci, determining the prevalence of this pathogen in non-native crayfish is vital to prioritize native crayfish populations for managed translocation. In the current study, 23 populations of invasive signal crayfish (Pacifastacus leniusculus) from the UK were tested for A. astaci presence using quantitative PCR. Altogether, 13 out of 23 (56·5%) populations were found to be infected, and pathogen prevalence within infected sites varied from 3 to 80%. Microsatellite pathogen genotyping revealed that at least one UK signal crayfish population was infected with the A. astaci genotype group B, known to include virulent strains. Based on recent crayfish distribution records and the average rate of signal crayfish population dispersal, we identified one native white-clawed crayfish (Austropotamobius pallipes) population predicted to come into contact with infected signal crayfish within 5 years. This population should be considered as a priority for translocation.

Oomycete Species Associated with Soybean Seedlings in North America-Part II: Diversity and Ecology in Relation to Environmental and Edaphic Factors.

Soybean (Glycine max (L.) Merr.) is produced across a vast swath of North America, with the greatest concentration in the Midwest. Root rot diseases and damping-off are a major concern for production, and the primary causal agents include oomycetes and fungi. In this study, we focused on examination of oomycete species distribution in this soybean production system and how environmental and soil (edaphic) factors correlate with oomycete community composition at early plant growth stages. Using a culture-based approach, 3,418 oomycete isolates were collected from 11 major soybean-producing states and most were identified to genus and species using the internal transcribed spacer region of the ribosomal DNA. Pythium was the predominant genus isolated and investigated in this study. An ecology approach was taken to understand the diversity and distribution of oomycete species across geographical locations of soybean production. Metadata associated with field sample locations were collected using geographical information systems. Operational taxonomic units (OTU) were used in this study to investigate diversity by location, with OTU being defined as isolate sequences with 97% identity to one another. The mean number of OTU ranged from 2.5 to 14 per field at the state level. Most OTU in this study, classified as Pythium clades, were present in each field in every state; however, major differences were observed in the relative abundance of each clade, which resulted in clustering of states in close proximity. Because there was similar community composition (presence or absence) but differences in OTU abundance by state, the ordination analysis did not show strong patterns of aggregation. Incorporation of 37 environmental and edaphic factors using vector-fitting and Mantel tests identified 15 factors that correlate with the community composition in this survey. Further investigation using redundancy analysis identified latitude, longitude, precipitation, and temperature as factors that contribute to the variability observed in community composition. Soil parameters such as clay content and electrical conductivity also affected distribution of oomycete species. The present study suggests that oomycete species composition across geographical locations of soybean production is affected by a combination of environmental and edaphic conditions. This knowledge provides the basis to understand the ecology and distribution of oomycete species, especially those able to cause diseases in soybean, providing cues to develop management strategies.

Oomycete Species Associated with Soybean Seedlings in North America-Part I: Identification and Pathogenicity Characterization.

Oomycete pathogens are commonly associated with soybean root rot and have been estimated to reduce soybean yields in the United States by 1.5 million tons on an annual basis. Limited information exists regarding the frequency and diversity of oomycete species across the major soybean-producing regions in North America. A survey was conducted across 11 major soybean-producing states in the United States and the province of Ontario, Canada. In 2011, 2,378 oomycete cultures were isolated from soybean seedling roots on a semiselective medium (CMA-PARPB) and were identified by sequencing of the internal transcribed spacer region of rDNA. Sequence results distinguished a total of 51 Pythium spp., three Phytophthora spp., three Phytopythium spp., and one Aphanomyces sp. in 2011, with Pythium sylvaticum (16%) and P. oopapillum (13%) being the most prevalent. In 2012, the survey was repeated, but, due to drought conditions across the sampling area, fewer total isolates (n = 1,038) were collected. Additionally, in 2012, a second semiselective medium (V8-RPBH) was included, which increased the Phytophthora spp. isolated from 0.7 to 7% of the total isolates. In 2012, 54 Pythium spp., seven Phytophthora spp., six Phytopythium spp., and one Pythiogeton sp. were recovered, with P. sylvaticum (14%) and P. heterothallicum (12%) being recovered most frequently. Pathogenicity and virulence were evaluated with representative isolates of each of the 84 species on soybean cv. Sloan. A seed-rot assay identified 13 and 11 pathogenic species, respectively, at 13 and 20°C. A seedling-root assay conducted at 20°C identified 43 species as pathogenic, having a significantly detrimental effect on the seedling roots as compared with the noninoculated control. A total of 15 species were pathogenic in both the seed and seedling assays. This study provides a comprehensive characterization of oomycete species present in soybean seedling roots in the major production areas in the United States and Ontario, Canada and provides a basis for disease management and breeding programs.

AFLP-PCR and RAPD-PCR evidences of the transmission of the pathogen Aphanomyces astaci (Oomycetes) to wild populations of European crayfish from the invasive crayfish species, Procambarus clarkii.

Aphanomyces astaci (Oomycetes) is responsible for the crayfish plague disease. This species is endemic of North America and five genotypes have been described using RAPD-PCR. The red swamp crayfish, Procambarus clarkii, is one of the most widely spread North American species and invasive in the world. However, no outbreaks on its specific genotype, i.e., genotype D, have ever been described in nature. We investigated three major series of crayfish plague outbreaks in indigenous crayfish populations of Austropotamobius pallipes, located in the areas of influence of P. clarkii. All samples collected tested positive for A. astaci using a rnDNA ITS-PCR test. We also performed an AFLP-PCR analysis on 19 isolates, and found that all isolates belong to genotype D. These isolates exhibited similar properties, i.e., adaptation to warm temperatures. We demonstrate, for the first time, the transmission of A. astaci genotype D to indigenous European populations of crayfish, and confirm that the properties of adaptation to warm water temperatures seem to be a specific character of genotype D. The results of this work emphasize once more the need of controlling invasive species and its trade, since they can carry harmful pathogens with specific adaptations or increased virulence in new environments.

A Sensitive Assay for Rapid Detection and Quantification of Aphanomyces euteiches in Soil.

Aphanomyces euteiches is a widespread oomycete pathogen causing root rot in a wide range of leguminous crops. Losses can reach up to 100% for pea culture and there is currently no registered pesticide for its control. Crop management remains the most efficient tool to control root rot, and avoidance of infested soil seems to be the optimal solution. A test was developed to identify fields suitable for pea crops, consisting of the determination of the inoculum potential of soil using baiting plants. A new rapid, specific, and sensitive molecular method is described allowing the quantification of less than 10 oospores per gram of soil. This challenge is achieved by a real-time polymerase chain reaction procedure targeting internal transcribed spacer 1 from the ribosomal DNA operons. A preliminary study based on typical soils from northwestern France demonstrated that the A. euteiches oospore density in soil is related to the inoculum potential. Furthermore, this method has proved sensitive enough to accurately study the influence of biotic factors that may govern the actual emergence of root rot.

Microsatellite markers for direct genotyping of the crayfish plague pathogen Aphanomyces astaci (Oomycetes) from infected host tissues.

Aphanomyces astaci is an invasive pathogenic oomycete responsible for the crayfish plague, a disease that has devastated European freshwater crayfish. So far, five genotype groups of this pathogen have been identified by applying random amplified polymorphic DNA analysis on axenic cultures. To allow genotyping of A. astaci in host tissue samples, we have developed co-dominant microsatellite markers for this pathogen, tested them on pure cultures of all genotype groups, and subsequently evaluated their use on tissues of (1) natural A. astaci carriers, i.e., North American crayfish species, and (2) A. astaci-infected indigenous European species from crayfish plague outbreaks. Out of over 200 potential loci containing simple sequence repeat (SSR) motifs identified by 454 pyrosequencing of SSR-enriched library, we tested 25 loci with highest number of repeats, and finally selected nine that allow unambiguous separation of all known RAPD-defined genotype groups of A. astaci from axenic cultures. Using these markers, we were able to characterize A. astaci strains from DNA isolates from infected crayfish tissues when crayfish had a moderate to high agent level according to quantitative PCR analyses. The results support the hypothesis that different North American crayfish hosts carry different genotype groups of the pathogen, and confirm that multiple genotype groups, including the one originally introduced to Europe in the 19th century, cause crayfish plague outbreaks in Central Europe. So far undocumented A. astaci genotype seems to have caused one of the analysed outbreaks from the Czech Republic. The newly developed culture-independent approach allowing direct genotyping of this pathogen in both axenic cultures and mixed genome samples opens new possibilities in studies of crayfish plague pathogen distribution, diversity and epidemiology.

Gold-nanoparticle based electrochemical DNA sensor for the detection of fish pathogen Aphanomyces invadans.

Epizootic ulcerative syndrome (EUS) is a devastating fish disease caused by the fungus, Aphanomyces invadans. Rapid diagnosis of EUS is needed to control and treat this highly invasive disease. The current diagnostic methods for EUS are labor intensive. We have developed a highly sensitive and specific electrochemical genosensor towards the 18S rRNA and internal transcribed spacer regions of A. invadans. Multiple layers of latex were synthesized with the help of polyelectrolytes, and labeled with gold nanoparticles to enhance sensitivity. The gold-latex spheres were functionalized with specific DNA probes. We describe here the novel application of this improved platform for detection of PCR product from real sample of A. invadans using a premix sandwich hybridization assay. The premix assay was easier, more specific and gave higher sensitivity of one log unit when compared to the conventional method of step-by-step hybridization. The limit of detection was 0.5 fM (4.99 zmol) of linear target DNA and 1 fM (10 amol) of PCR product. The binding positions of the probes to the PCR amplicons were optimized for efficient hybridization. Probes that hybridized close to the 5' or 3' terminus of the PCR amplicons gave the highest signal due to minimal steric hindrance for hybridization. The genosensor is highly suitable as a surveillance and diagnostic tool for EUS in the aquaculture industry.

Development and standardization of a monoclonal antibody-based rapid flow-through immunoassay for the detection of Aphanomyces invadans in the field.

A monoclonal antibody-based flow-through immunoassay (FTA) was developed using a nitrocellulose membrane placed on the top of adsorbent pads enclosed in a plastic cassette with a test zone at the center. The FTA could be completed within 10 min. Clear purple dots against a white background indicated the presence of Aphanomyces (A.) invadans. The FTA limit of detection was 7 µg/mL for A. invadans compared to 56 µg/mL for the immunodot. FTA and polymerase chain reaction (PCR) could detect A. invadans in fish tissue homogenates at a 10(-11) dilution compared to a 10(-8) dilution by immunodot. In fish suffering from natural cases of epizootic ulcerative syndrome (EUS) collected from Mangalore, India, FTA and PCR could detect A. invadans in 100% of the samples compared to 89.04% detected by immunodot. FTA reagents were stable and produced expected results for 4 months when stored at 4~8°C. This rapid test could serve as simple and cost-effective on-site screening tool to detect A. invadans in fish from EUS outbreak areas and in ports during the shipment of live or frozen fish.

Prevalence of the crayfish plague pathogen Aphanomyces astaci in populations of the signal crayfish Pacifastacus leniusculus in France: evaluating the threat to native crayfish.

Aphanomyces astaci, the crayfish plague pathogen, first appeared in Europe in the mid-19(th) century and is still responsible for mass mortalities of native European crayfish. The spread of this parasite across the continent is especially facilitated by invasive North American crayfish species that serve as its reservoir. In France, multiple cases of native crayfish mortalities have been suggested to be connected with the presence of the signal crayfish Pacifastacus leniusculus, which is highly abundant in the country. It shares similar habitats as the native white-clawed crayfish Austropotamobius pallipes and, when infected, the signal crayfish might therefore easily transmit the pathogen to the native species. We investigated the prevalence of A. astaci in French signal crayfish populations to evaluate the danger they represent to local populations of native crayfish. Over 500 individuals of Pacifastacus leniusculus from 45 French populations were analysed, plus several additional individuals of other non-indigenous crayfish species Orconectes limosus, O. immunis and Procambarus clarkii. Altogether, 20% of analysed signal crayfish tested positive for Aphanomyces astaci, and the pathogen was detected in more than half of the studied populations. Local prevalence varied significantly, ranging from 0% up to 80%, but wide confidence intervals suggest that the number of populations infected by A. astaci may be even higher than our results show. Analysis of several individuals of other introduced species revealed infections among two of these, O. immunis and P. clarkii. Our results confirm that the widespread signal crayfish serves as a key reservoir of Aphanomyces astaci in France and therefore represents a serious danger to native crayfish species, especially the white-clawed crayfish. The prevalence in other non-indigenous crayfish should also be investigated as they likely contribute to pathogen transmission in the country.

Temporal dynamics of spore release of the crayfish plague pathogen from its natural host, American spiny-cheek crayfish (Orconectes limosus), evaluated by transmission experiments.

The crayfish plague pathogen, Aphanomyces astaci, is one of the most serious threats to indigenous European crayfish species. The North American invasive spiny-cheek crayfish, Orconectes limosus, is an important source of this pathogen in central and western Europe. We evaluated potential changes in A. astaci spore release rate from infected individuals of this species by experiments investigating the pathogen transmission to susceptible noble crayfish, Astacus astacus. We filtered defined volumes of water regularly to quantify spore concentration, and sampled crayfish tissues at the end of the experiment. The filters and tissues were then tested for the presence of A. astaci DNA by species-specific quantitative PCR. Additionally, we tested the efficiency of horizontal transmission to apparently uninfected O. limosus. The experiments confirmed that A. astaci can be transmitted to susceptible crayfish during intermoult periods, and that the pathogen was more frequently detected in noble crayfish recipients than in American ones. The pathogen spore concentrations substantially varied in time, and significantly increased during moulting of infected hosts. Our study strengthens the evidence that although the likelihood of crayfish plague transmission by water transfer from localities with infected American crayfish might increase when these are moulting or dying, no time-periods can be proclaimed safe.

Timing and quantifying Aphanomyces astaci sporulation from the noble crayfish suffering from the crayfish plague.

Aphanomyces astaci sporulation is crucial for the spreading potential of this disease agent. For the first time, we are reporting timing and quantity of A. astaci spores released from noble crayfish (Astacus astacus) suffering from crayfish plague under practical aquatic conditions. We infected nine noble crayfish with A. astaci PsI-genotype and maintained them in individual 8L tanks. Spores (zoospores and cysts) were quantified from water samples (3 × 1 mL) taken every 12h over 10 d using A. astaci specific qPCR. A clear sporulation trend was found, together with a high individual spore estimate variation. The median spore counts from two days before death to 12h post mortem were from ~500 to ~2000 spores L(-1). A significant sporulation increase occurred after 24h post mortem (~12,000 spores L(-1)) and reached a peak after two days (~65,000 spores L(-1)) before declining to or below pre mortem levels from the fourth day. The single most sporulating crayfish released from ~75,000 to ~400,000 spores L(-1) during the mass sporulating period, yielding a maximum estimate of ~3,200,000 spores released from a single crayfish if we assume homogeneous spore distribution. The results confirm a mass A. astaci spore release from moribund and recently dead infected noble crayfish, with a sporulation peak one to three days post mortem. The acute crayfish mortality only three days after zoospore exposure confirm the lethal potential of the PsI-genotype. The powerful sporulation potential observed here may be one of the key virulence factors of this genotype.

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

Native European crayfish, such as Astacus leptodactylus, are threatened, among other factors, by the crayfish plague agent Aphanomyces astaci, dispersed by invasive North American crayfish. Two of these invaders, Pacifastacus leniusculus and Orconectes limosus, have extended their distribution in the River Danube catchment; the latter was detected for the first time in Romania in 2008. We monitored, at monthly intervals for over 2 yr, occurrence of native A. leptodactylus and invasive O. limosus at 6 sites on the Romanian Danube and checked for the invasive species in 4 of its tributaries. Between January 2009 and March 2011, the relative abundances of O. limosus steadily increased with time, while the native A. leptodactylus dramatically decreased in abundance. O. limosus expanded downstream at a rate of ca. 15 km yr-1; in August 2011, it was already present in the upper 105 km of the Romanian Danube. An agent-specific real-time PCR analyses demonstrated the presence of A. astaci DNA in at least 32% of the analysed invasive (n = 71) and 41% of the native (n = 49) crayfish coexisting in the Danube. Furthermore, A. astaci was also detected in A. leptodactylus captured about 70 km downstream of the O. limosus invasion front (at the time of sampling). Assuming a steady rate of expansion, O. limosus may invade the sensitive Danube delta area in the mid-2060s, even without long-distance dispersal. The crayfish plague agent, however, may reach the delta substantially earlier, through dispersal downstream among populations of native crayfish.

A comparative study of molecular diagnostic methods designed to detect the crayfish plague pathogen, Aphanomyces astaci.

Crayfish plague is the most important disease of freshwater crayfish with a significant impact on European species. We compared the analytical test sensitivity and specificity of three published PCR assays for the detection of Aphanomyces astaci, the causative agent of crayfish plague: a conventional PCR assay targeting the ITS region and two TaqMan(®) real time assays, targeting either the ITS region or the chitinase gene. We also tested a variation of the conventional assay, by changing one of the primers. Test specificity was assessed using DNA from a range of A. astaci strains and an array of closely related Oomycetes, host tissue and DNA from other organisms that may be present in a diagnostic sample. Sensitivity was assessed using genomic A. astaci DNA from mycelium and zoospores. All assays were found to be of good to excellent sensitivity with levels of detection ranging from 1 (real time assay targeting the ITS region), over 10 (conventional PCR) to 100 zoospores (real time assay targeting the chitinase gene). All three published assays were also specific for A. astaci and did not cross-react with any other test organisms included in this study. The tested variation of the conventional PCR assay with a changed forward primer led to amplification of some non A. astaci DNA. Advantages and disadvantages, including suitable application are discussed for each assay.

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

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 semi-nested 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.