Duration and method of fixation affects the sensitivity of a digoxygenin-labelled DNA probe in detecting Kudoa thyrsites in Atlantic salmon skeletal muscle

The effect of fixative and duration of fixation on the sensitivity of a non-radioactive in situ hybridisation (ISH) protocol to detect Kudoa thyrsites small subunit ribosomal deoxyribonucleic acid (DNA) was investigated. Strong ISH reactions were detected in 5-μm sections of paraffin-embedded Atlantic salmon muscle after fixation for 1 day in Davidson's solution (DS). Reactions were weak following 3 or 5 days fixation and absent after 17 or 28 days fixation. Strong ISH reactions were observed after 1, 3 or 5 days fixation in neutral buffered formalin (NBF). The reactions were weak after 17 days and weak to nonexistent after 28 days of fixation. Reactions were consistently strong after fixation in 95% ethanol for up to 28 days. Some mature spores reacted weakly or not at all by ISH. Parasite DNA was weakly amplified by polymerase chain reaction (PCR) from paraffin-embedded muscle after 1 day of fixation in DS but not after fixation for 3, 5, 17 or 28 days. Amplified DNA was detected after fixation in NBF for 1, 3 and 5 days, but not after 17 or 28 days. In contrast, PCR consistently amplified DNA from paraffin-embedded, ethanol-fixed muscle. Caution should be used in the choice of fixative and duration of fixation when preserving Atlantic salmon tissues for molecular diagnosis of K. thyrsites.