title: 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.
authors.affiliation: Norwegian Veterinary Institute, P.O. Box 750 Sentrum, , N-0106, Oslo, Norway. turhan.markussen@vetinst.no.
authors.forename: Turhan
authors.initials: T
authors.lastname: Markussen
authors.affiliation: Norwegian Veterinary Institute, P.O. Box 750 Sentrum, , N-0106, Oslo, Norway. celia.agusti@nmbu.no.|Present address: Institute of Food Safety and Infection Biology, Norwegian University of Life Sciences, P.O. Box 8146, , Dep., NO-0033, Oslo, Norway. celia.agusti@nmbu.no.
authors.forename: Celia
authors.initials: C
authors.lastname: Agusti
authors.affiliation: Institute of Marine Research, P.O. Box 1870, , Nordnes, 5817, Bergen, Norway. egil.karlsbakk@imr.no.
authors.forename: Egil
authors.initials: E
authors.lastname: Karlsbakk
authors.affiliation: Department of Biology, University of Bergen, Thormøhlensgt 55, N-5020, Bergen, Norway. are.nylund@bio.uib.no.
authors.forename: Are
authors.initials: A
authors.lastname: Nylund
authors.affiliation: Cermaq Group AS, Dronning Eufemias gate 16, N-0191, Oslo, Norway. oyvind.brevik@cermaq.com.
authors.forename: Øyvind
authors.initials: Ø
authors.lastname: Brevik
authors.affiliation: Norwegian Veterinary Institute, P.O. Box 750 Sentrum, , N-0106, Oslo, Norway. haakon.hansen@vetinst.no.
authors.forename: Haakon
authors.initials: H
authors.lastname: Hansen
mesh_terms: D000818:Animals; D016054:DNA, Protozoan / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D005393:Fish Diseases / Q000175:diagnosis* / Q000469:parasitology; D017403:In Situ Hybridization / Q000379:methods*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D055764:Myxozoa / Q000302:isolation & purification*; D015345:Oligonucleotide Probes / Q000235:genetics; D010273:Parasitic Diseases, Animal / Q000175:diagnosis* / Q000469:parasitology; D010274:Parasitology / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D020317:Salmo salar / Q000469:parasitology*; D012680:Sensitivity and Specificity; D014730:Veterinary Medicine / Q000379:methods
chemical_list: D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D015345:Oligonucleotide Probes; D012337:RNA, Ribosomal, 18S