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.