Fluorescent labelling of actinospores for determining the portals of entry into fish

A fluorescent dye, 5(6)-carboxyfluorescein diacetate, succinimidyl-ester (CFSE), was used for pre-labelling of actinospores to determine their portals of entry into fishes. Three types of actinospores, triactinomyxon (Myxobolus arcticus), raabeia (Myxobolus cultus) and aurantiactinomyxon (Thelohanellus hovorkai), were freshly isolated from the oligochaete hosts, and incubated with CFSE for 15 min. Myxosporean-free salmonid or cyprinid fishes were exposed to the CFSE-labelled actinospores for 30 min, and fixed with 2% paraformaldehyde. The fixed fins, skin and gills were examined for the presence of CFSE-labelled cells under a fluorescence microscope. Penetration of triactinomyxon and raabeia was mostly observed on the fins and skin. In contrast, aurantiactinomyxon penetrated mainly via the gills of common carp. Aurantiactinomyxon did not penetrate goldfish, which was non-susceptible to ?: hovorkai. However, penetration of triactinomyxon was noted in the epithelium of sockeye salmon, which had a low susceptibility to M. arcticus in Japan. This is the first report of a CFSE-labelling technique used for tracking infectious organisms in transmission experiments.