The crayfish plague, caused by the pathogen Aphanomyces astaci, is a pandemic disease endemic to North America that has been devastating susceptible crayfish populations in Europe since the 19th century. In Spain, this disease has decimated populations of the native crayfish species Austropotamobius pallipes due to introductions of North American crayfish, which act as vectors of the pathogen. To combat against these losses, several regional governments have established ex-situ breeding programs to restock wild populations of the species. In this study, we report on an outbreak of A. astaci that occurred in one of the most important A. pallipes aquaculture centers in Spain. Using a variety of detection methods, we analyzed affected crayfish and environmental samples from the facilities over a period of six months and determined that the outbreak was caused by two haplotypes of A. astaci, d1 and d2, which are both associated with the North American crayfish species Procambarus clarkii. To our knowledge, this is the first report of a two-haplotype coinfection of A. astaci outside the native range of this pathogen.
