warmer or cooler waters are less likely to be infected, and infection rates vary seasonally. Susceptibility Fish size, age, concentration of triactinomyxon spores, and water temperature all affect infection rates in fish, as does the species of the fish in question. The disease has the most impact on fish less than five months old because their skeletons have not ossified. This makes young fish more susceptible to deformities and provides M. cerebralis more cartilage on which to feed. In one study of seven species of many strains, brook trout and rainbow trout (except one strain) were far more heavily affected by M. cerebralis after two hours of exposure than other species were, while bull trout, Chinook salmon, brown trout, and Arctic grayling were least severely affected. While brown trout may harbor the parasite, they typically do not show any symptoms, and this species may have been M. cerebralis''' original host. This lack of symptoms in brown trout meant that the parasite was only discovered after nonnative rainbow trouts were introduced in Europe. Diagnosis The normally uniform trout cartilage is scarred with lesions in which M. cerebralis spores develop, weakening and deforming the connective tissues. Moderate or heavy clinical infection of fish with whirling disease can be presumptively diagnosed on the basis of changes in behavior and appearance about 35 to 80 days after initial infection, though "injury or deficiency in dietary tryptophan and ascorbic acid can evoke similar signs", so conclusive diagnosis may require finding myxospores in the fish's cartilage. In heavy infections, only examining cartilage microscopically may be necessary to find spores. In less severe infections, the most common test involves digestion of the cranial cartilage with the proteases pepsin and trypsin (pepsin-trypsin digest—PTD) before looking for spores. The head and other tissues can be further examined using histopathology to confirm whether the location and morphology of the spores matches what is known for M. cerebralis. Serological identification of spores in tissue sections using an antibody raised against the spores is also possible. Parasite identity can also be confirmed using polymerase chain reaction to amplify the 415 base pair 18S rRNA gene from M. cerebralis. Fish should be screened at the life stage most susceptible to the parasites, with particular focus on fish in aquaculture units. Impact Although originally a mild pathogen of Salmo trutta in central Europe and other salmonids in northeast Asia, the introduction of the rainbow