Abstract
The oomyceteAphanomyces astaciis the etiologic agent of crayfish plague, a disease that has seriously impacted the populations of European native crayfish species. The introduction of non-indigenous crayfish of North American origin and their wide distribution across Europe have largely contributed to spread of crayfish plague in areas populated by indigenous crayfish. TrackingA. astacigenotypes may thus be a useful tool for investigating the natural history of crayfish plague in its European range, as well as the sources and introduction pathways of the pathogen. In this study, we describe the development of real-time PCR TaqMan assays aiming to distinguish the five genotype groups ofA. astaci(A–E) previously defined by their distinct RAPD patterns. The method was evaluated using DNA extracts from pureA. astacicultures representing the known genotype groups, and fromA. astaci-positive crayfish clinical samples collected mostly during crayfish plague outbreaks that recently occurred in Central Italy and Czechia. The assays do not cross-react with each other, and those targeting genotype groups A, B, D, and E seem sufficiently specific to genotype the pathogen from infected crayfish in the areas invaded byA. astaci(particularly Europe). The unusualA. astacigenotype “SSR-Up” documented from crayfish plague outbreaks in Czechia and chronically infectedPontastacus leptodactylusin the Danube is detected by the group B real-time PCR. The assay originally developed to detect group C (one not yet documented from crayfish plague outbreaks) showed cross-reactivity withAphanomyces fennicus; theA. astacigenotype “rust1” described in the United States fromFaxonius rusticusis detected by that assay as well. Analyses of additional markers (such as sequencing of the nuclear internal transcribed spacer or mitochondrial ribosomal subunits) may complement such cases when the real-time PCR-based genotyping is not conclusive. Despite some limitations, the method is a robust tool for fast genotyping ofA. astacigenotype groups common in Europe, both during crayfish plague outbreaks and in latent infections.
Highlights
Crayfish plague is a disease of freshwater crayfish caused by the oomycete Aphanomyces astaci, which has been endangering the populations of indigenous crayfish throughout Europe and adjacent regions for over 150 years (Alderman, 1996; Holdich et al, 2009; OIE (World Organisation for Animal Health), 2019)
We describe the development and application of a new method based on real-time PCR as an alternative approach to currently available methods for the quick identification of common A. astaci genotype groups causing crayfish plague outbreaks in Europe
Molecular typing of A. astaci strains, which differentiates distinct genotype groups, contributes to a better understanding of the relationship between this pathogen and its host taxa
Summary
Crayfish plague is a disease of freshwater crayfish caused by the oomycete Aphanomyces astaci, which has been endangering the populations of indigenous crayfish throughout Europe and adjacent regions for over 150 years (Alderman, 1996; Holdich et al, 2009; OIE (World Organisation for Animal Health), 2019). North American crayfish coevolved with this oomycete and only succumb to crayfish plague under particular conditions (e.g., Unestam, 1969, 1972; DiéguezUribeondo and Söderhäll, 1993; Diéguez-Uribeondo et al, 1995), but can act as carriers of the infection [e.g., Persson and Söderhäll, 1983; Diéguez-Uribeondo and Söderhäll, 1993; Jussila et al, 2015; OIE (World Organisation for Animal Health), 2019]. With the development of suitable molecular methods, chronic infections in populations of crayfish species that are generally considered susceptible but had not experienced mass mortalities or other symptoms of acute crayfish plague were documented (see a review in Svoboda et al, 2017)
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