In situ Hybridization for Detection of the Microsporidian Parasite Glugea plecoglossi by Using Rainbow Trout as an Experimental Infection Model.

Abstract

Early stages of Glugea plecoglossi (Microspora) are difficult to detect by normal light microscopy. In situ hybridization (ISH) protocol with DIG-labeled oligonucleotide probes designed from SSUrRNA of G. plecoglossi was established with the aim of detecting all stages. All developmental stages of G. plecoglossi in xenomas could be detected by ISH. First, we aimed to evaluate the suitability of rainbow trout as an experimental animal in place of ayu for G. plecoglossi. Immersion challenge tests showed that the susceptibility and location of G. plecoglossi did not differ between ayu and rainbow trout. Cyst forming sites were different among challenged trout by the three methods (intubation, immersion and intraperitoneal injection). In the intestinal epithelium of rainbow trout (Oncorhynchus mykiss) at 5 min post-intubation of G. plecoglossi spores, we found possible initial stages, which were positive in ISH and negative in Uvitex 2B staining. This finding suggests that G. plecoglossi spores discharging the polar tube pierced the gut epithelial cells, and injected the infective sporoplasm. The present study showed that ISH is a promising tool for studying transmission modes of G. plecoglossi in fish and that rainbow trout is a useful replacement host to identify the pathobiological characteristics of G. plecoglossi.