Tracing the route of Sphaerospora truttae from the entry locus to the target organ of the host, Salmo salar L., using an optimized and specific in situ hybridization technique

Sphaerospora truttae is an important pathogen of Atlantic salmon parr in Scottish aquaculture. To trace the early development of S. truttae and to overcome the common problem of detecting low numbers of cryptic, early myxosporean stages, a DNA-based approach was applied in this study. Specific primers were designed for S. truttae from Atlantic salmon, based on 18S rDNA sequences, obtained from isolated myxosporean spores. These were 5' biotin-labelled and used in an optimized and rapid in situ hybridization (ISH) protocol, which provided a strong and specific signal of the parasite within host tissue sections and, at the same time, minimized structural damage to tissues due to processing. This methodology provided a reliable tool enabling the detection of S. truttae stages down to single cell level. Using ISH the epithelium of the gills was identified as the predominant entry locus of the parasite. By 3 days after infection S. truttae had penetrated the vascular epithelia and thereafter proliferated in the blood for at least 10 days before exiting the vascular system through capillary walls. From day 12 post-infection onwards, the kidney, as well as the spleen and the liver, were invaded. Numbers of S. truttae invading the kidney (37.3%) differed little from numbers found in the spleen (35.3%) and the liver (27.4%). The latter organs represented a dead end in the development of S. truttae as all stages in these organs degenerated and sporogony was found to take place exclusively inside the renal tubules. Early sporogonic stages were found from day 25 post-infection but mature spores only developed after at least 15 days of proliferation within the tubules.