Detection of the myxosporean parasite Parvicapsula pseudobranchicola in Atlantic salmon (Salmo salar L.) using in situ hybridization (ISH).

Abstract

BACKGROUND: Parvicapsula pseudobranchicola is a marine myxosporean parasite infecting farmed Atlantic salmon (Salmo salar). A major site for the parasite is the pseudobranch, which may be destroyed in heavily infected fish. Parvicapsulosis may be associated with significant mortality, although the main effect of infections seems to be runting. In situ hybridization (ISH) is, in the absence of specific antibodies, the preferred method for the detection of cell- and tissue tropisms of myxozoans in the early phases of infection of the host, and provides information about the possible association between the pathogen and pathology. A positive diagnosis of parvicapsulosis is based on histopathology and PCR. The aim of the present work was to develop a specific, sensitive and robust ISH assay for the detection of P. pseudobranchicola in tissues. METHODS: The ISH method was designed to specifically target P. pseudobranchicola 18S rDNA/rRNA using a locked nucleic acid (LNA) modified oligonucleotide probe. The method was tested on paraffin embedded P. pseudobranchicola infected pseudobranchs. The infections were confirmed by light microscopy revealing the presence of typical P. pseudobranchicola trophozoites and spores, and the presence of parasite was confirmed with real-time RT-PCR. RESULTS: Specific regions stained by ISH overlapped well with the parasitized and degenerated regions in neighbouring HE stained sections. No staining was observed in pseudobranchs of Atlantic salmon which had been held in P. pseudobranchicola-free water. CONCLUSIONS: We report here the development of a sensitive ISH assay for the detection of P. pseudobranchicola in paraffin embedded tissue. The technique will be valuable in the study of host entry, early proliferation, pre-spore development, pathology and tissue tropism in Atlantic salmon.