title: Microsatellite markers for direct genotyping of the crayfish plague pathogen Aphanomyces astaci (Oomycetes) from infected host tissues.
abstract: 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.
authors.affiliation: Laboratoire Ecologie et Biologie des Interactions, Equipe: Ecologie, Evolution, Symbiose, Université de Poitiers, UMR CNRS 7267, 40 av. du Recteur Pineau, F-86022 Poitiers Cedex, France. Electronic address: frederic.grandjean@univ-poitiers.fr.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Grandjean
authors.affiliation: Norwegian Veterinary Institute, N-0106 Oslo, Norway; Microbial Evolution Research Group (MERG), Department of Biosciences, University of Oslo, N-0316 Oslo, Norway.
authors.forename: Trude
authors.initials: T
authors.lastname: Vrålstad
authors.affiliation: Departamenta de Micologia, Real Jardín Botánico CSIC, Plaza Murillo 2, 28014 Madrid, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Diéguez-Uribeondo
authors.affiliation: University of Zagreb, Faculty of Science, Division of Biology, Department of Zoology, Rooseveltov trg 6, 10000 Zagreb, Croatia.
authors.forename: Mišel
authors.initials: M
authors.lastname: Jelić
authors.affiliation: Laboratoire Ecologie et Biologie des Interactions, Equipe: Ecologie, Evolution, Symbiose, Université de Poitiers, UMR CNRS 7267, 40 av. du Recteur Pineau, F-86022 Poitiers Cedex, France.
authors.forename: Joa
authors.initials: J
authors.lastname: Mangombi
authors.affiliation: Laboratoire Ecologie et Biologie des Interactions, Equipe: Ecologie, Evolution, Symbiose, Université de Poitiers, UMR CNRS 7267, 40 av. du Recteur Pineau, F-86022 Poitiers Cedex, France.
authors.forename: Carine
authors.initials: C
authors.lastname: Delaunay
authors.affiliation: Laboratoire Ecologie et Biologie des Interactions, Equipe: Ecologie, Evolution, Symbiose, Université de Poitiers, UMR CNRS 7267, 40 av. du Recteur Pineau, F-86022 Poitiers Cedex, France; Charles University in Prague, Faculty of Science, Department of Ecology, Viničná 7, CZ-12844 Prague, Czech Republic.
authors.forename: Lenka
authors.initials: L
authors.lastname: Filipová
authors.affiliation: Departamenta de Micologia, Real Jardín Botánico CSIC, Plaza Murillo 2, 28014 Madrid, Spain.
authors.forename: Svetlana
authors.initials: S
authors.lastname: Rezinciuc
authors.affiliation: Charles University in Prague, Faculty of Science, Department of Ecology, Viničná 7, CZ-12844 Prague, Czech Republic.
authors.forename: Eva
authors.initials: E
authors.lastname: Kozubíková-Balcarová
authors.affiliation: Laboratoire Signalisation et Transports Ioniques Membranaires STIM ERL 7368, 1 rue George Bonnet, F 86022 Poitiers, France.
authors.forename: Daniel
authors.initials: D
authors.lastname: Guyonnet
authors.affiliation: OIE Reference Laboratory for Crayfish Plague, Finnish Food Safety Authority Evira, Neulaniementie 4, FI-70210 Kuopio, Finland.
authors.forename: Satu
authors.initials: S
authors.lastname: Viljamaa-Dirks
authors.affiliation: Charles University in Prague, Faculty of Science, Department of Ecology, Viničná 7, CZ-12844 Prague, Czech Republic.
authors.forename: Adam
authors.initials: A
authors.lastname: Petrusek
mesh_terms: D000818:Animals; D044744:Aphanomyces / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D003400:Astacoidea / Q000469:parasitology*; D005060:Europe; D014644:Genetic Variation; D005838:Genotype; D018895:Microsatellite Repeats / Q000235:genetics*; D019105:Random Amplified Polymorphic DNA Technique
keywords: Aphanomyces astaci; Crayfish mass mortalities; Crayfish plague; Genotyping; Invasive crayfish; Microsatellites